Technical Field
-
The present invention relates to a therapeutic agent
for cibophobia, which comprises, as an active ingredient,
a substance that suppresses expression or function of orphan
GPCR expressed in hypothalamus. Specifically, the present
invention relates to a therapeutic agent for cibophobia,
which comprises, as an active ingredient, antisense nucleic
acid of GPCR mRNA, an expression vector containing the nucleic
acid or a host cell transfected with the expression vector,
or a therapeutic agent for cibophobia, which comprises, as
an active ingredient, a substance having an antagonist
activity to GPCR. Furthermore, the present invention
relates to a coexpression system of a GPCR and a G protein
and a screening method for a therapeutically active compound
for cibophobia using the same. The present invention
moreover relates to a therapeutic agent for a
lifestyle-related disease, which comprises, as an active
ingredient, a substance that enhances the expression or
function of GPCR. In detail, the present invention relates
to a therapeutic agent for a lifestyle-related disease, which
comprises, as an active ingredient, a substance having an
agonist activity to GPCR. Furthermore, the present
invention relates to a coexpression system of a GPCR and a
G protein and a screening method for a therapeutically active
compound for a lifestyle-related disease using the same.
Background Art
-
Due to westernization of the eating habits, increase
of social stress and the like in recent years, the number
of patients with obesity and accompanying lifestyle-related
diseases, particularly type II diabetes, has been increasing
dramatically. For the therapy of these cases, exercise
therapy and diet therapy are performed first. When the
weight control is insufficient even by these treatments, drug
therapy is performed. In doing so, a therapeutic agent which
is superior in controlling food intake, body weight and blood
glucose, and which is safe has been desired.
-
On the other hand, stress society of the present age
combined with epidemic of an easy diet has brought a rapid
increase of psychogenic eating disorder, such as cibophobia,
in adolescent women. While it is indispensable to solve
psychological problems to treat these diseases fundamentally,
drug therapy may be performed to forcibly control feeding
behavior as a supportive therapy. A therapeutic agent in
this case is also requested to be able to promote eating while
controlling body weight and glucose level.
-
Eating is mainly controlled by the central nervous
system, and many nervous systems ruling over instinctive
behaviors of human, such as appetite and the like, are located
particularly in hypothalamus. In fact, when hypothalamus
ventromedial nucleus of rat is damaged, it causes overeating
and obesity, whereas when hypothalamus lateral nucleus is
damaged, feeding behavior is not taken. In addition,
localization of receptors of leptin and neuropeptides (e.g.,
neuropeptide Y (NPY)), which are involved in eating control,
has been shown heretofore, which makes it clear that
hypothalamus is an important organ for feeding behavior.
-
It has become clear that receptors of physiologically
active substances in the central nervous system including
hypothalamus, particularly G protein-coupled receptor
(GPCR), correlates with feeding behavior. For example, it
is known that knockout (KO) mouse with serotonin 5-THT2C
receptor suffers from chronic overeating. In addition,
melanocortin 4 receptor antagonist increases food intake and,
on the contrary, NPY Y5 receptor antagonist suppresses food
intake.
-
Thus, stimulation of the nervous system in hypothalamus
is considered to influence the feeding behavior, and
substitute operation of signal transduction through GPCR,
which is expressed in hypothalamus, by the use of a small
compound meets the above-mentioned object of controlling
food intake, body weight, glucose level and the like.
However, a drug having such an action mechanism has not been
marketed at present, and development of such a pharmaceutical
agent has been highly desired.
-
It is therefore an object of the present invention to
provide a means for regulating feeding behavior by allowing
an external factor, particularly a factor that suppresses
or promotes expression or function of GPCR involved in the
feeding behavior, to function, thereby treating a
lifestyle-related disease mainly caused by overeating or
obesity, or cibophobia. It is another object of the present
invention to provide a compound having a controlling effect
on eating disorder such as overeating and apocleisis, obesity
or the like, and a screening method for such a compound.
Disclosure of the Invention
-
To achieve the above-mentioned objects, the present
inventors first examined a gene encoding a receptor expressed
in hypothalamus, and, as a result, found a certain orphan
GPCR (hereinafter to be referred to as GPRC5D) gene is
expressed in hypothalamus of obese model mouse. Next, the
present inventors administered an antisense oligo DNA of mRNA
encoding this receptor to obese model mouse to inhibit its
expression. As a result, food intake was in fact increased
and blood glucose level was elevated. Therefrom it has been
clarified that GPRC5D is a receptor involved in the signal
transduction which negatively regulates the feeding behavior,
and therefore, a substance inhibiting expression or function
of this receptor shows a therapeutic effect on eating
disorders such as cibophobia. In contrast, a substance
enhancing expression or function of this receptor should show
a therapeutic effect on lifestyle-related diseases including
type II diabetes caused by overeating, obesity and the like.
Thus, the present inventors constructed a series of
coexpression systems of GPRC5D and various G proteins, and
developed a method for screening for a compound having a
therapeutic activity against cibophobia or
lifestyle-related diseases by searching for an agonist or
an antagonist to GPRC5D using the system, which resulted in
the completion of the present invention.
-
Accordingly, the present invention provides a
therapeutic agent for cibophobia, which comprises, as an
active ingredient, a substance that suppresses expression
or function of GPRC5D, a GPCR expressed in hypothalamus. As
a substance capable of specifically suppressing the
expression of GPRC5D, an antisense nucleic acid of mRNA
encoding GPRC5D can be preferably mentioned. In this case,
the antisense nucleic acid can be provided not only as it
is but in the form of an expression vector encoding said
nucleic acid or a host cell into which said expression vector
has been introduced. In addition, as a substance capable of
specifically suppressing the function of GPRC5D, an
antagonist to said receptor can be mentioned.
-
The present invention further provides a therapeutic
agent for lifestyle-related diseases, which comprises, as
an active ingredient, a substance enhancing expression or
function of GPRC5D. As a substance capable of specifically
enhancing the function of GPRC5D, a physiological ligand and
an agonist to this receptor can be mentioned.
-
Therefore, another aspect of the present invention
provides a screening method for a substance having a
therapeutic activity against cibophobia or
lifestyle-related diseases, which comprises screening for
an antagonist or an agonist to GPRC5D. This method comprises
comparing a GDP/GTP exchange reaction of a G protein or the
activity of an effector the G protein acts upon, in the
presence and absence of the test substance, in a series of
receptor - G protein coexpression systems obtained by
constructing a constitution unit for a receptor-binding
region of each family of Gα, wherein one constitution unit
comprises a system comprising, as essential elements, at
least a lipid bilayer membrane comprising GPRC5D or an
equivalent thereof, and a polypeptide comprising at least
a receptor-binding region of a G protein α subunit
(hereinafter to be also referred to as Gα) belonging to a
certain family and a guanine nucleotide-binding region of
any G protein α subunit.
-
Accordingly, the present invention also provides a
screening system for a substance having a therapeutic
activity against cibophobia or a lifestyle-related disease,
which comprises the above-mentioned series of receptor-G
protein coexpression systems.
-
The present invention moreover provides a therapeutic
agent for cibophobia or a therapeutic agent for a
lifestyle-related disease, comprising, as an active
ingredient, a substance having a therapeutic activity
against cibophobia or a lifestyle-related disease, which is
obtained by the above-mentioned screening system or
screening method.
-
Further characteristics and advantages of the present
invention will become clear from the disclosure of "Best Mode
for Embodying the Invention" below.
Brief Description of the Drawings
-
- Fig. 1 shows the effect of administration of antisense
DNA of GPRC5D on food intake of obese mouse (immediately after
administration - 48 hours later), wherein black columns show
food consumption of control DNA administration groups, white
columns show that of antisense DNA administration groups
(average ± standard deviation, control DNA administration
groups n=4, antisense DNA administration groups n=4), and
* in the Figure indicates the presence of a significant
difference between the both groups (p<0.05, Student's
t-test).
- Fig. 2 shows the effect of administration of antisense
DNA of GPRC5D on blood glucose level of obese mouse
(immediately after administration - 48 hours later), wherein
black columns show blood glucose level of control DNA
administration groups, white columns show that of antisense
DNA administration groups (average ± standard deviation,
control DNA administration groups n=4, antisense DNA
administration groups n=4), and * in the Figure indicates
the presence of a significant difference between the both
groups (p<0.05, Student's t-test).
- Fig. 3 shows variation in expression of GPRC5D gene in
normal mouse and obese mouse in satiation state and fasting
state, wherein black columns show ratio of GPRC5D/GAPDH in
the satiation state, white columns show that in the fasting
state (average ± standard deviation, n=3 in each group).
- Fig. 4 shows cAMP concentrations in extract from CHO-K1
cell in which GPRC5D was transiently expressed alone or on
fusing with various Gα proteins, wherein mock shows CHO-K1
cells transfected with pcDNA3.1(+), GPRC5D shows CHO-K1
cells transfected with pcC5D, GPRC5D-Gs shows CHO-K1 cells
transfected with pcC5D/His/GαS2, GPRC5D-Gi shows CHO-K1
cells transfected with pcC5D/His/Gαi2 and GPRC5D-Gq shows
CHO-K1 cells transfected with pcC5D/His/Gα16.
-
Best Mode for Embodying the Invention
-
The present invention provides a therapeutic agent for
cibophobia, which comprises as an active ingredient a
substance that suppresses the expression or function of
GPRC5D, a GPCR expressed in the hypothalamus.
-
"GPRC5D" is one of human GPCR proteins consisting of
the amino acid sequence shown by SEQ ID NO:2, which has been
newly discovered by homology search on EST database using
a set of human GPCR amino acid sequence categorized into the
5th group of metabotrophic glutamate receptor-like family
(family C) (Brauner-Osborne et al., Biochim. Biophys. Acta,
1518(3): 237-48(2001); registered in GeneBank under
accession numbers: AF209923, XM006896 and NM018654).
However, physiological functions, physiological ligands and
coupling G protein (α subunit) subtypes, etc. of GPRC5D have
remained unclear. The present inventors have independently
found that this receptor gene is expressed in hypothalamus
of the obese model mouse and conducted further investigations
based on this finding. As a result, as described above, the
present inventors have identified this protein as a membrane
receptor involved in stimulation of the feeding center.
-
It is known that GPCR homologs corresponding to human
GPRC5D exist in other mammals [e.g., homolog mGprc5d (SEQ
ID NO:4) which has about 82% identity with human GPRC5D at
the amino acid level was discovered in mouse (Brauner-Osborne
et al., ibid. registered in GenBank under accession number:
AF218809). Hereinafter a simple reference to "GPRC5D" is to
be understood as collectively referring to human GPRC5D and
its mouse homolog.]. Accordingly, the therapeutic agent for
cibophobia of the present invention is intended for use to
eating disorders not only in humans but also in other mammals.
Since social environment of the present age is a stressful
environment for animals like livestocks and pets, the present
invention is useful for the application to the field of
veterinary medicine as well.
-
The therapeutic agent for cibophobia of the present
invention comprises, as an active ingredient, a substance
that suppresses the expression or function of GPRC5D. The
term "expression", as used herein, refers to a state wherein
a receptor protein is produced and functionally located on
the cell membrane. Accordingly, the "substance that
suppresses the expression" may act at any stage, such as at
the gene transcription level, post-transcription regulation
level, translation-into-protein level, post-translational
modification level, membrane transport level and protein
folding level. On the other hand, the "substance that
suppresses the function" refers to a substance that acts on
a receptor once functionally located on the cell membrane,
and that does not cause a shift of the equilibration between
the active and inactive forms at least toward the active side.
-
Examples of substances that suppress the expression
of GPRC5D include transcriptional suppressors, RNA
polymerase inhibitors, RNA-decomposing enzymes, protein
synthesis inhibitors, proteases, and protein denaturants;
to minimize the adverse effects on other genes and proteins
that are expressed in cells, it is important that the
substance should be capable of specifically acting on the
target molecule. Accordingly, a preferred embodiment of a
substance that suppresses the expression of GPRC5D is an
antisense nucleic acid of the GPRC5D mRNA (consisting of the
base sequence shown by SEQ ID NO:1 (indicates only ORF) or
SEQ ID NO:3) or its initial transcription product. The term
"antisense nucleic acid" refers to a nucleic acid that
consists of a base sequence capable of hybridizing to target
mRNA (initial transcription product) under the physiological
conditions of cells that express the target mRNA (initial
transcription product), and that is capable of inhibiting
the translation into the polypeptide encoded by the target
mRNA (initial transcription product) under the hybridized
state. The kind of antisense nucleic acid may be DNA or RNA
and may be a DNA/RNA chimera. Additionally, because natural
form of antisense nucleic acids have their phosphate di-ester
linkage decomposed easily by the nuclease present in cells,
the antisense nucleic acid of the present invention can also
be synthesized using modified nucleotides such as of the
thiophosphate type (phosphate bond P=O replaced with P=S)
or 2'-O-methyl type, which types are stable to the nuclease.
Other important requirements for designing an antisense
nucleic acid include increasing the water solubility and cell
membrane permeability; these goals can also be achieved by
improving the dosage form such as through the use of liposome
or microspheres.
-
The length of the antisense nucleic acid of the present
invention is not particularly limited, as long as the
antisense nucleic acid is capable of specifically
hybridizing to the GPRC5D mRNA or its initial transcription
product, and the antisense nucleic acid may be a sequence
comprising a sequence of about 15 bases at the shortest in
length or complementary to the entire sequence of the mRNA
(initial transcription product) at the longest. From the
viewpoint of ease of synthesis, antigenicity concern, and
other aspects, there may be mentioned, for example,
oligonucleotides consisting of preferably about 15 to about
30 bases. When the antisense nucleic acid is an about 25mer
oligo DNA, the base sequence capable of hybridizing to the
GPRC5D mRNA under physiological conditions may be any one,
as long as it possesses about 80% identity or more, depending
on the base composition of the target sequence.
-
The target sequence for the antisense nucleic acid of
the present invention is not particularly limited, as long
as it is a sequence such that the translation into GPRC5D
protein or a functional fragment thereof is inhibited as a
result of hybridization with the antisense nucleic acid, and
may be the entire sequence or a partial sequence of the GPRC5D
mRNA or may be the intron portion of the initial transcription
product. However, when using an oligonucleotide as the
antisense nucleic acid, it is desirable that the target
sequence should be located between the 5'-terminus of the
GPRC5D mRNA and the C-terminus of the coding region (the
region shown by base numbers 1-1035 in the base sequence shown
by SEQ ID NO:1 or base numbers 1-1047 in the base sequence
shown by SEQ ID NO:3). Preferably, the target sequence is
located on region between the 5'-terminus and the N-terminus
side of the coding region, with greatest preference given
to a base sequence in the vicinity of the initiation codon
(base numbers 148-150 in the base sequence shown by SEQ ID
NO:3). Additionally, it is preferable that the target
sequence should be selected such that an antisense nucleic
acid complementary thereto does not form a secondary
structure such as a hairpin structure.
-
Furthermore, the antisense nucleic acid of the present
invention may be capable of not only hybridizing to the GPRC5D
mRNA or its initial transcription product to inhibit the
translation, but also binding to the GPRC5D gene, a
double-stranded DNA, to form a triple-strand (triplex) to
inhibit the transcription into mRNA.
-
Another preferred embodiment of a substance that
suppresses GPRC5D expression is a ribozyme capable of
specifically cleaving within the coding region of the GPRC5D
mRNA or its initial transcription product (the base sequence
shown by base numbers 1-1035 in the base sequence shown by
SEQ ID NO:1 or base numbers 148-1047 in the base sequence
shown by SEQ ID NO:3) (including the intron portion in case
of the initial transcription product). The term "ribozyme"
refers to an RNA having an enzyme activity to cleave nucleic
acid. Since it has recently been shown that oligo DNA having
the base sequence of the enzyme activity site also possesses
nucleic acid cleavage activity, the term ribozyme is used
herein to include DNA, as long as it possesses
sequence-specific nucleic acid cleavage activity. The most
widely applicable ribozyme is self-splicing RNA found in
infectious RNA of viroids, virusoids, etc.; such ribozymes
include the hammerhead type and the hairpin type. The
hammerhead type exhibits enzyme activity with about 40 bases,
and is capable of specifically cleaving the target mRNA alone
by rendering several bases at each end (about 10 bases in
total) adjacent to the hammerhead structure complementary
to the desired cleavage site of the mRNA. This type of
ribozymes is also advantageous in that they do not attack
genomic DNA because their substrate is RNA alone. When the
GPRC5D mRNA by itself takes a double-stranded structure, it
is possible to render the target sequence single-stranded
by using a hybrid ribozyme resulting from the linking of an
RNA motif from viral nucleic acid that is capable of
specifically binding to RNA helicase [Proc. Natl. Acad. Sci.
USA, 98(10) : 5572-5577 (2001)]. Furthermore, when using the
ribozyme in the form of an expression vector containing DNA
that encodes the ribozyme, the ribozyme may be a hybrid
ribozyme resulting from the further linking of a sequence
of modified tRNA to promote the transfer to cytoplasm [Nucleic
Acids Res., 29(13): 2780-2788 (2001)].
-
Another embodiment of a substance that suppresses the
expression of GPRC5D is a double-stranded oligo RNA that is
complementary to a partial sequence in the coding region of
the GPRC5D mRNA or its initial transcription product
(including the intron portion in the case of the initial
transcription product). What is called RNA interference
(RNAi), a phenomenon wherein upon intracellular introduction
of a short double-stranded RNA, an mRNA complementary to that
RNA is decomposed, has long been known to occur in nematodes,
insects, plants, and other organisms. Since this phenomena
has recently been found to occur in animal cells as well
[Nature, 411(6836): 494-498 (2001)], RNAi is drawing
attention for its potential as an alternative to ribozyme.
-
The antisense oligonucleotide and ribozyme of the
present invention can be prepared by determining the target
sequence for the mRNA or its initial transcription product
on the basis of the GPRC5D cDNA sequence or genomic DNA
sequence, and synthesizing a complementary sequence using
a commercially available DNA/RNA autosynthesizer (Applied
Biosystems, Beckman, etc.). A double-stranded oligo RNA
possessing RNAi activity can be prepared by synthesizing a
sense strand and an antisense strand respectively using a
DNA/RNA autosynthesizer, denaturing each strand in the
appropriate annealing buffer at about 90°C to about 95°C for
about 1 minute, and subsequently annealing them at about 30°C
to about 70°C for about 1 to about 8 hours. Additionally,
a longer double-stranded polynucleotide can be prepared by
synthesizing complementary oligonucleotide strands in
alternative overlaps, annealing them, and subsequently
subjecting them to ligation with ligase.
-
A preferred embodiment of a substance that suppresses
the functional expression of GPRC5D at the
post-translational level is an antibody against GPRC5D or
a fragment thereof. This antibody may be a polyclonal
antibody or monoclonal antibody, and can be prepared by a
well-known immunological technique. Any fragment of the
anti-GPRC5D antibody serves for the purpose, as long as it
has an antigen-binding site (CDR) for GPRC5D, and is
exemplified by Fab, F(ab')2, ScFv, minibody, etc.
-
For example, a polyclonal antibody can be obtained by
giving GPRC5D protein or a fragment thereof [may be prepared
as a complex cross-linked with a carrier protein such as
bovine serum albumin or KLH (Keyhole Limpet Hemocyanin), if
necessary] as the antigen, along with a commercially
available adjuvant (e.g., complete or incomplete Freund's
adjuvant), to an animal by subcutaneous or intraperitoneal
administration about 2 to 4 times at intervals of 2 to 3 weeks
(the antibody titer of serum separated from drawn blood
determined by a commonly known antigen-antibody reaction,
and its elevation confirmed in advance), collecting whole
blood about 3 to about 10 days after final immunization, and
purifying the antiserum. Animals to be administered with the
antigen include mammals such as rats, mice, rabbits, goat,
guinea pigs and hamsters.
-
A monoclonal antibody can also be prepared by a cell
fusion method (e.g., Takeshi Watanabe, saibouyugouhou no
genri to monokuronaru kotai no sakusei, Akira Taniuchi and
Toshitada Takahashi, eds., "monokuronaru kotai to gan - kiso
to rinsho-", pp. 2-14, Science Forum Publishing, 1985). For
example, a mouse is given this factor, along with a
commercially available adjuvant, 2 to 4 times by subcutaneous
or intraperitoneal administration, its spleen or lymph node
is collected about 3 days after final administration, and
leukocytes are separated. These leukocytes are fused with
myeloma cells (e.g., NS-1, P3X63Ag8, etc.) to yield a
hybridoma that produces a monoclonal antibody against this
factor. The cell fusion may be achieved by the PEG method
[J. Immunol. Methods, 81(2): 223-228 (1985)] or the voltage
pulsation method [Hybridoma, 7(6): 627-633 (1988)]. A
hybridoma that produces the desired monoclonal antibody can
be selected by detecting in the culture supernatant an
antibody that specifically binds to an antigen using
well-known EIA, RIA, or the like. Cultivation of a hybridoma
that produces a monoclonal antibody can be conducted in vitro,
or in vivo in mice or rats, preferably in ascites fluid of
mouse, and the resulting antibody can be obtained from a
hybridoma culture supernatant or animal ascites fluid,
respectively.
-
However, in view of therapeutic effect and safety in
humans, the anti-GPRC5D antibody of the present invention
is preferably a chimeric antibody between a human and another
animal (e.g., mice etc.), more preferably a humanized
antibody. The term "chimeric antibody", as used herein,
refers to an antibody having a variable region (V region)
from an immunized animal and a constant region (C region)
from a human; "humanized antibody" refers to an antibody
wherein all regions except CDR have been replaced with a human
antibody. A chimeric antibody or a humanized antibody can,
for example, be obtained by cutting out a sequence that
encodes a V region or CDR from the gene for a mouse monoclonal
antibody prepared in the same manner as above, cloning a
chimeric gene resulting from fusion with DNA that encodes
a C region of an antibody from human myeloma into an
appropriate expression vector, and introducing the vector
to an appropriate host cell to express the chimeric gene.
-
Another preferred embodiment of a substance that
suppresses the functional expression of GPRC5D at the
post-translational level is an oligonucleotide that
specifically binds to GPRC5D and inhibits its functional
expression, i.e., aptamer. An aptamer for GPRC5D can, for
example, be obtained by the procedure shown below. First,
oligonucleotides (e.g., about 60 bases) are randomly
synthesized using a DNA/RNA autosynthesizer to obtain a pool
of oligonucleotides. Next, an oligonucleotide that binds to
the protein of interest, i.e., GPRC5D is separated using an
affinity column. The separated oligonucleotide is amplified
by PCR and again screened through the aforementioned
selection process. By repeating this process in about five
cycles or more, an aptamer showing high affinity for GPRC5D
can be selected.
-
A therapeutic agent for cibophobia comprising as an
active ingredient a substance that suppresses the expression
of GPRC5D is not capable of exhibiting its therapeutic
activity unless it is incorporated in cells of the target
tissue (i.e., hypothalamus); its active ingredient, nucleic
acid or protein molecule, is not easily absorbable in cells
and in addition is likely to undergo rapid decomposition in
the body. Additionally, because the uptake of these
molecules is usually carried out by endocytosis, they are
likely to undergo decomposition by lysosome enzyme.
Accordingly, it is important to design a drug delivery system
(DDS) wherein a substance that suppresses the expression of
GPRC5D is delivered to hypothalamic cells in a stable state
so as to increase cell membrane permeability and to promote
drug release from lysosome/endosome. For example, in the
case of an oligo nucleic acid molecule such as an antisense
oligonucleotide, it is possible to improve the stability to
nuclease, intracellular transfer, and release from
lysosome/endosome by chemical modifications such as PNA
resulting from the replacement of the phosphate and sugar
portions with peptide bonds, and oligonucleotides having a
morphine backbone in place of a phosphate backbone, as well
as nucleic acids with their phosphate linkage or sugar portion
(2' position, 3' position, etc.) modified as described above;
these modified oligo nucleic acids can easily be prepared
using a DNA/RNA autosynthesizer.
-
On the other hand, cell membrane permeability can also
be increased by coupling an accessory group such as
poly-L-lysine, avidin, cholesterol or phospholipid to an
oligonucleotide or antibody molecule.
-
Furthermore, it is also possible to prepare an
oligonucleotide or antibody molecule as incorporated in a
cationic liposome. By incorporating in a liposome, the
active ingredient is protected against decomposition by
nuclease and protease, and is incorporated in cells by
endocytosis with the cationic surface of the liposome
membrane binding to negatively charged molecules on the cell
surface. Cationic liposome can, for example, be prepared by
mixing a cationic lipid, such as DOTMA, DDAB or DMRIE, and
DOPE, a neutral lipid capable of membrane fusion. Because
nucleic acid and proteins are polyanionic, they easily form
complexes when mixed with cationic liposomes. Additionally,
it is possible to achieve cell-specific targeting by
inserting in the liposome membrane an antibody or ligand for
a cell surface molecule that is expressed specifically in
hypothalamus cells. For example, the anti-GPRC5D antibody
itself may be inserted in the liposome membrane.
-
Additionally, to protect the liposome incorporated by
endocytosis against decomposition by lysosome enzyme, it is
also preferable to use a pH-sensitive liposome (at acidic
pH levels, the membrane becomes unstable and its contents
are released from endosomic vesicles to cytoplasm before
fusion with lysosome) or a liposome fused with Sendai virus
wherein the viral RNA has been completely fragmented by
ultraviolet irradiation etc. (the endocytosis pathway
avoided by means of the membrane fusion capability of Sendai
virus).
-
The therapeutic agent for cibophobia of the present
invention designed in a dosage form as described above can
be administered orally or parenterally by dissolving or
suspending in an appropriate sterile vehicle. Examples of
parenteral administration route include, for example, but
are not limited to, systemic administrations such as
intravenous, intra-arterial, intramuscular,
intraperitoneal and intratracheal administrations, and
local administration in the vicinity of the hypothalamus.
Preferably, there may be mentioned local administration to
the lateral ventricle.
-
The dosage of the therapeutic agent for cibophobia of
the present invention varies depending on the kind of active
ingredient, molecule size, administration route, severity
of disease, animal species of administration subject, drug
acceptability of administration subject, body weight, age,
etc., and normally ranges from about 0. 0008 to about 2.5 mg/kg,
preferably about 0.008 to about 0.025 mg/kg, based on the
amount of active ingredient per day for each adult; such a
dose may be administered at a time or in divided portions.
-
When the substance that suppresses the expression of
GPRC5D is a nucleic acid molecule like an antisense nucleic
acid, ribozyme or aptamer (hereinafter also referred to as
effective nucleic acid molecule), the therapeutic agent for
cibophobia of the present invention may comprise as an active
ingredient an expression vector that encodes the effective
nucleic acid molecule. With regard to the expression vector,
oligonucleotide or polynucleotide that encodes the
aforementioned effective nucleic acid molecule must be
functionally linked to a promoter capable of exhibiting
promoter activity in hypothalamus cells of the recipient
mammal, or arranged at a position such that the
oligonucleotide or polynucleotide is capable of turning to
a form functionally linked to the promoter under particular
conditions in hypothalamus cells of the administration
animmal. Any promoter can be used, as long as it is capable
of working in hypothalamus cells of the recipient mammal;
such promoters include, for example, viral promoters such
as the SV40-derived early promoter, cytomegalovirus LTR,
Rous sarcoma virus LTR, MoMuLV-derived LTR and
adenovirus-derived early promoter, and mammal constitutive
protein gene promoters such as the β-actin gene promoter,
PGK gene promoter and transferrin gene promoter. The wording
"arranged at a position such that the oligonucleotide or
polynucleotide is capable of turning to a form functionally
linked to a promoter under particular conditions" means that,
for example, the promoter and the oligo (poly) nucleotide that
encodes an effective nucleic acid molecule are split by two
recombinase recognition sequences arranged in the same
direction, which are separated by a spacer sequence long
enough to prevent expression of the effective nucleic acid
molecule from the promoter, such that the spacer sequence
is cleaved out in the presence of a recombinase that
specifically recognizes the recognition sequence, thereby
the polynucleotide that encodes the effective nucleic acid
molecule is functionally linked to the promoter, as described
in more detail below.
-
The expression vector of the present invention
contains a transcription termination signal, i.e., a
terminator region, preferably at downstream of an
oligo(poly)nucleotide that encodes the effective nucleic
acid molecule. Furthermore, the expression vector of the
present invention may further contain selection marker genes
for transformant selection (genes that confer resistance to
such drugs as tetracycline, ampicillin, kanamycin,
hygromycin and phosphinoslysine, genes that complement
auxotrophic mutation, etc.). When the expression vector has
a spacer sequence between recombinase recognition sequences
as described above, the selection marker gene may also be
arranged in the spacer sequence.
-
The vector used for the expression vector of the
present invention is not particularly limitated; examples
of vectors that are suitable for administration to mammals
such as humans include viral vectors such as retrovirus,
adenovirus, adeno-associated virus, herpes virus, vaccinia
virus, pox virus, polio virus, Sindbis virus and Sendai virus.
Adenovirus is advantageous in a number of features, including
extremely high gene introduction efficiency and the
capability of being introduced into non-dividing cells. It
should be noted, however, that because the incorporation of
the introduced gene into the host chromosome is extremely
rare, this gene expression is transient and usually only lasts
for about 4 weeks. In view of the persistence of the
therapeutic effect, it is also preferable to use an
adeno-associated virus, which is of relatively high gene
introduction efficiency, which can be introduced to
non-dividing cells as well, and which can be incorporated
into chromosomes via an inverted terminal repeat sequence
(ITR).
-
Effective nucleic acid molecules such as of antisense
nucleic acids and ribozyme are in essence foreign substances;
their constitutive and excess expression is highly toxic to
the host animal introduced with gene and may cause adverse
reactions. Accordingly, in a preferred embodiment of the
present invention, the expression vector is capable of
allowing an effective nucleic acid molecule to express
time-specifically and/or hypothalamus cell-specifically to
avoid the adverse effects of the excess expression of the
effective nucleic acid molecule at an unwanted time and/or
unwanted site. As a first example of such a vector, there
may be mentioned a vector containing an
oligo (poly) nucleotide that encodes an effective nucleic acid
molecule linked functionally to a promoter derived from a
gene specifically expressed in hypothalamus cells of the
administration animal. There may be mentioned, for example,
the native promoter of the GPRC5D gene.
-
As a second example of the time-specific and
hypothalamus-specific expression vector of the present
invention, there may be mentioned a vector containing an
oligo (poly) nucleotide that encodes an effective nucleic acid
molecule functionally linked to an inducible promoter which
regulate an expressionby an exogenous substance in trans.
For example, when using the metallothionein-1 gene promoter
as the inducible promoter, the expression of the effective
nucleic acid molecule can be induced
hypothalamus-specifically at any time by locally
administering an inducer such as a heavy metal, e.g., gold,
zinc and cadmium, a steroid, e.g., dexamethasone, an
alkylating agent, a chelating agent or a cytokine to the
hypothalamus at the desired time.
-
Another preferred example of the time-specific and
hypothalamus-specific expression vector of the present
invention is a vector having the promoter and the
oligo (poly) nucleotide that encodes an effective nucleic acid
molecule which are split by two recombinase recognition
sequences arranged in the same direction, wherein the
recombinase recognition sequences are separated by a spacer
sequence sufficiently long to prevent the expression of the
effective nucleic acid molecule from the promoter. Solely
introducing the vector in hypothalamus cells does not ensure
that the promoter directs the transcription of the effective
nucleic acid molecule. However, provided that a recombinase
that specifically recognizes the recognition sequence is
locally administered to the hypothalamus at the desired time,
or an expression vector containing a polynucleotide that
encodes recombinase is locally administered to express the
recombinase in hypothalamus cells, homologous recombination
via the recombinase occurs between the recognition
sequences; as a result, the spacer sequence is cleaved out
and the oligo(poly)nucleotide that encodes the effective
nucleic acid molecule is functionally linked to the promoter,
resulting in the hypothalamus-specific expression of the
effective nucleic acid molecule at the desired time.
-
It is desirable that the recombinase recognition
sequence used in the aforementioned vector should be a
heterologous recombinase recognition sequence that is not
recognized by endogenous recombinase so as to prevent the
recombination by the recombinase present in the recipient.
It is desirable, therefore, that the recombinase that act
on the vector in trans should also be a heterologous
recombinase. Preferred examples of such combinations of
heterologous recombinase and the recombinase recognition
sequence include, but are not limited to, a combination of
Escherichia coli bacteriophage P1-derived Cre recombinase
and the lox P sequence, and a combination of yeast-derived
Flp recombinase and the frt sequence.
-
Discovered in a bacteriophage, Cre recombinase is
known to work in the specific DNA recombination reaction,
not only in prokaryotic cells but also in animal cells which
are eukaryotic cells and animal viruses. When two lox P
sequences are present on the same DNA molecule in the same
direction, Cre recombinase cleaves out the DNA sequence
between the sequences to allow them to form a cyclic molecule
(cleavage reaction). On the other hand, in cases where two
lox P sequences are present on different DNA molecules one
of which is cyclic DNA, the cyclic DNA is inserted to the
other DNA molecule via the lox P sequence (insertion reaction)
[J. Mol. Biol., 150: 467-486 (1981) ; J. Biol. Chem., 259:
1509-1514 (1984); Proc. Natl. Acad. Sci. USA, 81: 1026-1029
(1984)]. Example cleavage reactions are reported in
cultured animal cells [Nucleic Acids Res., 17: 147-161
(1989); Gene, 181: 207-212 (1996)], animal viruses [Proc.
Natl. Acad. Sci. USA, 85: 5166-5170 (1988); J. Virol., 69:
4600-4606 (1995); Nucleic Acids Res., 23: 3816-3821 (1995)],
transgenic mice [Proc. Natl. Acad. Sci. USA, 89: 6232-6236
(1992); Proc. Natl. Acad. Sci. USA, 89: 6861-6865 (1992);
Cell, 73: 1155-1164 (1993); Science, 265:103-106 (1994)],
etc.
-
As the promoter for the time-specific and
hypothalamus-specific expression vector of the present
invention, which is based on the interaction of a
recombinase/recombinase recognition sequence, there may
preferably be used a virus-derived promoter or a mammal
constitutive protein gene promoter to ensure the expression
at the desired time and site.
-
Administration of the therapeutic agent for cibophobia
of the present invention comprising an expression vector that
encodes an effective nucleic acid molecule as an active
ingredient is carried out by either the ex vivo method, in
which nerve cells of the animal to be treated are taken out
from the body, cultured, then returned to the body by
introduction, and the in vivo method, in which the vector
is introduced by directly administering it to the recipient's
body. In case of the ex vivo method, introduction of the
vector into the target cell can be carried out by the
microinjection method, calcium phosphate co-precipitation
method, PEG method, electroporation method, etc. In case of
the in vivo method, the viral vector is administered in the
form of an injection or the like intravenously,
intra-arterially, subcutaneously, intracutaneously,
intramuscularly, intraperitoneally or the like.
Alternatively, administering a vector by intravenous
injection etc. may pose a problem with the production of a
neutralizing antibody against the viral vector; however, it
is possible to mitigate the adverse effects of the presence
of the antibody by locally injecting the vector in the
vicinity of the hypothalamus, where the target cell is present,
e.g., in the lateral ventricle (in situ method).
-
Additionally, when using a non-viral vector as the
expression vector that encodes an effective nucleic acid
molecule, introduction of the expression vector can be
carried out by use of a high molecular carrier such as a
poly-L-lysine-nucleic acid complex or liposome
encapsulation as described above with respect to dosage forms
of therapeutic drugs comprising the effective nucleic acid
molecule itself as an active ingredient. Alternatively, it
is also possible to introduce the vector directly to the
target cell using the particle gun method.
-
When recombinase itself is locally administered as the
trans-acting substance in the use of a vector based on
recombinase/recombinase recognition sequence interaction,
recombinase, for example, may be injected to the hypothalamus
on dissolving or suspending in an appropriate sterile vehicle
(e.g., artificial cerebrospinal fluid etc.). On the other
hand, when a recombinase expression vector is locally
administered to the hypothalamus as the trans-acting
substance, the recombinase expression vector may be any
vector, as long as it possesses an expression cassette having
the recombinase-encoding polynucleotide functionally linked
to a promoter capable of exhibiting promoter activity in
hypothalamus cells of the administration subject. When the
promoter used is a constitutive promoter, it is desirable
that the vector administered to the hypothalamus for
preventing the expression of recombinase at unwanted times
should be a vector that rarely undergoes incorporation in
the host cell chromosome, e.g., adenovirus. However, when
using an adenovirus vector, the transient expression of
recombinase persists for about 4 weeks at most; if the
treatment is prolonged, a second or third administration will
be necessary. As another approach to expressing a
recombinase at the desired time, there may be mentioned the
use of an inducible promoter like the metallothionein gene
promoter. In this case, viral vectors of high integration
efficiency such as retrovirus can be used.
-
When the substance that suppresses the expression of
GPRC5D is a nucleic acid molecule like an antisense nucleic
acid, ribozyme or aptamer, the therapeutic agent for
cibophobia of the present invention may contain as an active
ingredient a host cell containing an expression vector that
encodes an effective nucleic acid molecule as described above.
As examples of useful host cells, there may be mentioned
autologous cells taken out as target cells from the recipient
in the aforementioned ex vivo introduction method for an
expression vector, nerve cells taken out from allogenic (e.g.,
stillborn fetuses, brain death patients, etc., in case of
humans) or heterologous (non-human mammals such as swine and
simian, in case of humans) individuals, or nerve cells
obtained by culturing and differentiating such nerve stem
cells or ES cells. Because the central nervous system is the
organ/tissue where rejection is most unlikely, even
heterologous cells can be allowed to take using a small amount
of immunosuppressant in combination.
-
In another embodiment, it is possible to transform a
resident bacterium in the nasal cavity, throat, oral cavity,
intestine, or the like of the recipient animal as the host
cell, with an expression vector that encodes an effective
nucleic acid molecule by a conventional method, and to deliver
the thus-obtained transformant to a site of the recipient
where the host cell normally occurs. In recent years, a route
other than the blood-brain barrier route has been
investigated via which a drug is transferred from the nose
directly to the cerebrospinal fluid for delivery to the brain;
the use of a nasal cavity resident bacterium suffices that
objective.
-
The dosage of the therapeutic agent for cibophobia of
the present invention comprising, as an active ingredient,
an expression vector that encodes an effective nucleic acid
molecule or a host cell harboring the expression vector
varies depending on the kind of active ingredient, molecule
size, promoter activity, administration route, severity of
disease, animal species of administration subject, drug
acceptability of administration subject, body weight, age,
etc., and is preferably at a level that causes expression
of an effective nucleic acid molecule in an amount equivalent
to an appropriate dosage of a therapeutic drug comprising
the effective nucleic acid molecule itself as an active
ingredient in the body of an animal that has received a vector
or host cell, and is exemplified by about 2 to about 20 µg/kg,
preferably about 5 to about 10 µg/kg based on the amount of
vector per day for each adult.
-
Because GPRC5D is a membrane receptor protein that
mediates signal transduction for negatively regulating food
consumption, food intake behavior can be suppressed by
enhancing the expression of this receptor. Accordingly, the
present invention also provides a therapeutic agent for a
lifestyle-related disease comprising a substance that
enhances the expression of GPRC5D as an active ingredient.
In general, the "lifestyle-related disease" is defined as
a group of diseases whrein lifestyles such as dietary habits,
exercise habits, resting, smoking and drinking are
responsible for the onset and progress thereof. The same
term, as used herein, specifically refers to "a group of
diseases wherein a therapeutic effect can be achieved by
adjusting food intake to reduce it," typically exemplified
by diabetes, obesity, hyperlipidemia, hyperuricemia, etc.
Patients often have two or more of these diseases at a time.
-
Examples of substances that enhance the expression of
GPRC5D include trans-acting factors capable of promoting RNA
transcription from the GPRC5D gene, factors capable of
promoting splicing or mRNA transfer to cytoplasm, factors
that suppress mRNA decomposition, factors capable of
promoting binding of ribosome to mRNA, factors that suppress
the decomposition of the GPRC5D protein, and factors that
promote the transport of the GPRC5D protein to the membrane;
as preferred examples of more directly acting specific
substances, there may be mentioned the GPRC5D protein or an
equivalent thereof, an expression vector containing a
nucleic acid that encodes the GPRC5D, or a host cell harboring
the expression vector.
-
The "GPRC5D protein" as used herein is a protein
consisting of the amino acid sequence shown by SEQ ID NO:2
or SEQ ID NO:4; "an equivalent of GPRC5D protein" refers to
a polypeptide consisting of an amino acid sequence resulting
from the substitution, deletion, insertion, addition or
modification of 1 or more (preferably 1 to 50, more preferably
1 to 30, still more preferably 1 to 10, and most preferably
1 to 5) amino acids in the amino acid sequence shown by SEQ
ID NO:2 or SEQ ID NO:4, that exhibits a ligand-receptor
interaction equivalent to that of a protein consisting of
the amino acid sequence shown by SEQ ID NO: 2 or SEQ ID NO:4,
and that couples with Gα to promote the GDP-GTP exchange
reaction of the subunit or, being outside the range of this
definition, a protein derived from a different mammal, i.e.
orthlog, which is encoded by a gene having the same molecular
evolution origin as human or mouse GPRC5D gene. Accordingly,
the therapeutic agent for a lifestyle-related disease of the
present invention is intended for use to treat diabetes,
obesity, hyperlipidemia, hyperuricemia, etc., not only in
humans or mice but also in other mammals. Because the number
of animals suffering from diseases like lifestyle-related
diseases, such as obesity due to excess feeding and a lack
of exercise, has been increasing with the recent pet animal
boom, the remedy of the present invention is very useful in
the field of veterinary medicine as well.
-
The GPRC5D protein or an equivalent thereof can be
isolated from a membrane-containing fraction derived from
the hypothalamus tissue of humans or mice, or other mammals
such as bovine, swine, simian or rat by affinity
chromatography with the anti-GPRC5D antibody.
Alternatively, a DNA clone isolated from a cDNA library or
genomic library derived from the tissue with the GPRC5D cDNA
clone as a probe can be cloned into an appropriate expression
vector, introduced to the host cell, expressed, and purified
from the membrane-containing fraction of the cell culture
by affinity chromatography with the anti-GPRC5D antibody or
His-tag, GST-tag or the like. The equivalent may be
partially introduced a mutation by an artificial treatment
such as site-directed mutagenesis based on the GPRC5D cDNA
sequence (the base sequence shown by base numbers 1-1035 in
the base sequence shown by SEQ ID NO: 1 or base numbers 148-1047
in the base sequence shown by SEQ ID NO:3). Conservative
amino acid substitution is well known; those skilled in the
art can introduce a mutation as appropriate in the GPRC5D
protein, as long as the receptor characteristics of GPRC5D
remain unchanged. However, because the ligand binding
domain and preferably the extracellular loop to which an
inverse agonist is capable of binding, and the N-terminal
strand must be conserved to high extents, it is desirable
that a mutation should not be introduced in such regions.
-
A therapeutic agent for a lifestyle-related disease
comprising the GPRC5D protein or an equivalent thereof as
an active ingredient can be modified to increase its cell
membrane permeability by coupling an accessory group such
as poly-L-lysine, avidin, cholesterol or phospholipid
component as described above with respect to a therapeutic
agent for cibophobia comprising the anti-GPRC5D antibody as
an active ingredient. Alternatively, this therapeutic agent
can also be prepared by encapsulating the GPRC5D protein or
an equivalent thereof into a cationic liposome. Because
proteins are poly-anionic, the protein easily forms a complex
when mixed with a cationic liposome. Additionally, it is
also possible to achieve cell-specific targeting by
incorporating into the liposome membrane an antibody or
ligand for a cell surface molecule specifically expressed
in hypothalamus cells. For example, it is also possible to
incorporate the anti-GPRC5D antibody (preferably an antibody
not having antagonist activity or inverse agonist activity)
to the liposome membrane.
-
A therapeutic agent for a lifestyle-related disease
comprisng the GPRC5D protein or an equivalent thereof as an
active ingredient can be administered orally or parenterally
on dissolving or suspending in an appropriate sterile vehicle.
Examples of parenteral administration route include, for
example, but are not limited to, systemic administrations
such as intravenous, intra-arterial, intramuscular,
intraperitoneal and intratracheal administrations, and
local administration in the vicinity of the hypothalamus.
Preferably, there may be mentioned local administration to
the lateral ventricle.
-
The dosage of the present therapeutic agent for a
lifestyle-related disease varies depending on the
administration route, severity of disease, animal species
of administration subject, drug acceptability of
administration subject, body weight, age, etc., and normally
ranges from about 0.0008 to about 2.5 mg/kg, preferably about
0.008 to about 0.025 mg/kg based on the amount of active
ingredient per day for each adult; such a dose may be
administered at a time or in divided portions.
-
When the substance that enhances the expression of
GPRC5D is the GPRC5D protein or an equivalent thereof, the
therapeutic agent for a lifestyle-related disease of the
present invention may be an expression vector containing a
nucleic acid that encodes such a polypeptide, or may be a
host cell harboring the expression vector. The expression
vector and host cell used here may be identical to those used
for the aforementioned cibophobia remedy. Furthermore,
regarding the administration route and dosage for these
lifestyle-related disease remedies, those exemplified above
with respect to cibophobia remedies can be used preferably.
-
The present invention also provides a therapeutic
agent for cibophobia comprising as an active ingredient a
substance that suppresses the function of GPRC5D expressed
on the cell membrane of hypothalamus cells, or a therapeutic
agent for a lifestyle-related disease comprising as an active
ingredient a substance that promotes such function. These
therapeutic agents can be obtained by screening for
substances that exhibit agonist activity, antagonist
activity or inverse agonist activity to GPRC5D. Accordingly,
the present invention also provides at a time a screening
method for a substance that suppresses or promotes the
function of GPRC5D, and a screening system for the same
method.
-
The term "agonist activity", as used herein, refers
to a property by which the substance in question specifically
binds to the GPRC5D receptor and causes a shift of the
equilibration between the active and inactive forms of GPRC5D
toward the active side. Accordingly, substances having
agonist activity include physiological ligands for GPRC5D,
as well as what is called full agonists and partial agonists.
The term "antagonist activity" refers to a property by which
the substance in question competitively binds to the
ligand-binding site of GPRC5D but has no or almost no effect
on the equilibration between the active and inactive forms.
Accordingly, substances having antagonist activity are
understood to be what is called neutral antagonists, and not
to include inverse agonists. The term "inverse agonist
activity" refers to a property by which the substance in
question binds to any site of GPRC5D and causes a shift of
the equilibration between the active and inactive forms of
GPRC5D toward the inactive side. The simple term "ligand"
as used herein, is understood to include all physiological
ligands, agonists, antagonists and inverse agonists.
-
The screening system of the present invention is a
series of receptor-G protein co-expression systems obtained
by constructing a constituent unit for the receptor-binding
region of each Gα family (i.e., Gαs, Gαi, Gαq), which
constituent unit consists of a lipid bilayer membrane
containing the GPRC5D protein or an equivalent thereof, and
a polypeptide comprising at least the receptor-binding
region of a Gα belonging to a family and the guanine
nucleotide-binding region of any Gα, as essential member
constituents. The GPRC5D protein or an equivalent thereof
is identical to that mentioned above as an active ingredient
of the aforementioned therapeutic agent for a
lifestyle-related disease. Although the lipid bilayer
membrane retaining the GPRC5D protein or an equivalent
thereof may be of any origin, as long as the receptor protein
is allowed to take the essential steric structure on the
membrane, it is preferably exemplified by fractions
containing the cell membrane of eukaryotic cells such as human,
bovine, swine, simian, mouse, rat or other mammal cells, and
insect cells, e.g., intact cells, cell homogenates, or cell
membrane fractions fractionated from these homogenates by
centrifugation etc.. However, an artificial lipid bilayer
membrane prepared by a conventional method from a solution
of various lipids, e.g., phosphatidylcholine,
phosphatidylserine, and cholesterol, mixed at an appropriate
ratio, preferably a ratio close to abundance ratios in the
cell membranes of eukaryotic cells such as mammal cells and
insect cells, can also be used preferably in an embodiment
of the present invention.
-
Gα (Gαs), belonging to the Gs family, promotes the
activity of adenylate cyclase as the effector, and is
exemplified by Gαs-1-Gαs-4 and Golf. Gα (Gαi), belonging to the
Gi family, suppresses the activity of adenylate cyclase as
the effector, and is exemplified by Gαi-1-Gαi-3 and Gz. Gα
(Gαq), belonging to the Gq family, promotes the activity of
phospholipase C as the effector, and is exemplified by Gαq
and G16. The Gαs polypeptide, Gαi polypeptide and Gαq
polypeptide of the present screening system need to have a
region involved in the binding to its own GPCR (RB region)
and a region involved in the binding to any guanine nucleotide
of Gα (GB region). Results of X-ray crystallographic
analysis of Gα have shown that GPCR-binding region is located
in the vicinity of the C-terminus whereas the GB region is
a region homologous to the nucleotide-binding site of the
ras protein (from the N-terminus side: amino acid motives
called the P box, G' box, G box, and G" box, the leader of
the αE helix in a highly helical domain, αF helix, etc.).
When a physiological ligand or agonist to GPRC5D binds to
the receptor, the Gα activation domain of the receptor and
the Gα RB region that couples with the receptor interact with
each other to produce a conformational change in Gα, resulting
in the dissociation of GDP from the GB region and quick binding
of GTP. Gα-GTP acts on the effector to promote or suppress
its activity. On the other hand, binding an inverse agonist
inactivates the Gα active domain due to a conformational
change in the receptor, resulting in a decreased active Gα-GTP
level and inhibition of its action on the effector. Here,
provided that a GTP analogue that does not undergo hydrolysis
by the GTPase activity of Gα, such as 35S-labeled GTPγS, has
been added to the system in place of GTP, it is possible to
evaluate the effects of the test substance on the GDP-GTP
exchange reaction in Gα by determining and comparing the
radioactivity bound to the membrane in the presence and
absence of the test substance, and to screen for substances
that possess ligand activity for GPRC5D. Hence, provided
that the radioactivity has increased in the presence of a
test substance, the test substance can be judged to possess
agonist activity to GPRC5D and hence therapeutic activity
for lifestyle-related disease. Conversely, provided that
the radioactivity has decreased, the test substance can be
judged to possess inverse agonist activity to GPRC5D and hence
therapeutic activity for cibophobia.
-
Once ligands for GPRC5D are screened for, a family that
couples with the receptor is elucidated; subsequent
screening can be conducted using only a system containing
a Gα polypeptide belonging to the family as a member
constituent. The results described below of constitutive
activation of GPRC5D using a receptor-Gα fusion protein
expression system strongly suggest that the G protein α
subunit capable of coupling with GPRC5D may be Gαs.
Accordingly, the present invention also provides a screening
method for ligands for the receptor characterized in that
the GDP-GTP exchange reaction of the Gα or the activity of
the effector that interacts with the Gα is compared in the
presence and absence of the test substance in a co-expression
system of a G protein α subunit (preferably Gαs) capable of
coupling with GPRC5D and the receptor.
-
The activity of a ligand for GPRC5D can also be
determined with the action of a Gα polypeptide on the effector
as an index. In this case, the screening system of the
present invention must further comprise as another
constituent a lipid bilayer membrane containing the effector,
in addition to the GPRC5D protein or an equivalent thereof.
Additionally, the Gα polypeptide must further comprise a
region for interaction with the effector. Because the Gα
members belonging to the individual families differ with
respect to the kind of effector or the direction of action,
it is preferable that all Gα polypeptides share the effector
interaction region, rather than each has its own effector
interaction region. Accordingly, in the present screening
system, at least two kinds of Gα polypeptides are chimeric
polypeptides containing the effector interaction region of
a Gα belonging to another family. For example, when using
phospholipase C as the effector, the Gαq polypeptide may be
a native one; however, the Gαs polypeptide and Gαi polypeptide
must be chimeric polypeptides wherein the effector
interaction region has been replaced with that of Gαq. As
the simplest example of a chimeric polypeptide containing
the effector interaction region of a Gα belonging to another
family, there may be mentioned a chimeric polypeptide wherein
about 5 amino acids at the C-terminus of a Gα belonging to
another family (i.e., RB region) have been replaced with its
own C-terminal sequence.
-
In the present screening system, when three kinds of
Gα polypeptides contain an effector interaction region of
Gαs or Gαi, a lipid bilayer membrane containing adenylate
cyclase is used as the effector. On the other hand, when the
Gα polypeptide contains the effector interaction region of
Gαq, a lipid bilayer membrane containing phospholipase C must
be used as the effector.
-
In a screening system comprising adenylate cyclase
(hereinafter also referred to as AC) as the effector, the
action of a Gα polypeptide on the effector can be evaluated
by directly determining the AC activity. The AC activity can
be determined using any commonly known technique; examples
of useful methods include, but are not limited to, a method
comprising adding ATP to an AC-containing membrane fraction,
and determining the resulting cAMP content by competitive
immunoassay using an anti-cAMP antibody in the presence of
cAMP labeled with a RI (e.g., 125I), an enzyme (e.g., alkaline
phosphatase, peroxidase, etc.), a fluorescent substance
(e.g., FITC, rhodamine, etc.), or the like, and a method
comprising adding [α-32P] ATP to an AC-containing membrane,
separating the resulting [32P] cAMP using an alumina column
etc., and subsequently determining the radioactivity thereof.
When the Gα polypeptide contains the effector interaction
region of Gαs, and determining and comparing the AC activity
in the presence and absence of a test substance, provided
that the AC activity has increased in the presence of the
test substance, the test substance can be judged to possess
agonist activity to GPRC5D and hence therapeutic activity
for lifestyle-related disease. Conversely, provided that
the AC activity has decreased, the test substance can be
judged to possess inverse agonist activity to GPRC5D and hence
therapeutic activity for cibophobia. On the other hand, when
the Gα polypeptide contains the effector interaction region
of Gα, provided that the AC activity has increased in the
presence of a test substance, the test substance can be judged
to possess inverse agonist activity to GPRC5D and hence
therapeutic activity for cibophobia. Conversely, provided
that the AC activity has decreased, the test substance can
be judged to possess agonist activity to GPRC5D and hence
therapeutic activity for lifestyle-related disease.
-
When using an intact eukaryotic cell as the screening
system, the action of a Gα polypeptide on AC can also be
evaluated by determining the intracellular cAMP content, or
labeling the cell with [3H] adenine, and determining the
radioactivity of resulting [3H] cAMP. Although the
intracellular cAMP content can be determined by incubating
cells in the presence and absence of the test substance for
an appropriate time, subsequently disrupting the cells, and
subjecting the thus-obtained extract by the aforementioned
competitive immunoassay, any other commonly known method can
be used.
-
In another embodiment, the cAMP content may be
evaluated by determining the amount of expression of reporter
gene under the control of the cAMP-response element (CRE).
The expression vector used here is described in detail below,
and is outlined below. The intracellular cAMP content is
determined by culturing a eukaryotic cell incorporating a
vector containing an expression cassette with a DNA that
encodes the reporter protein linked downstream of a
CRE-containing promoter, in the presence and absence of the
test substance for an appropriate time, disrupting the cells,
and measuring and comparing the expression of the reporter
gene in the thus-obtained extract using a commonly known
technique.
-
Accordingly, when the Gα polypeptide contains the
effector interaction region of Gas, provided that the
intracellular cAMP content (or the amount of expression of
reporter gene under the control of CRE) has increased in the
presence of a test substance, the test substance can be judged
to possess agonist activity to GPRC5D and hence therapeutic
activity for lifestyle-related disease. Conversely,
provided that the cAMP content (or the amount of expression
of reporter gene) has decreased, the test substance can be
judged to possess inverse agonist activity to GPRC5D and hence
therapeutic activity for cibophobia. On the other hand, when
the Gα polypeptide contains the effector interaction region
of Gαi, provided that the intracellular cAMP content (or the
amount of expression of reporter gene under the control of
CRE) has increased in the presence of a test substance, the
test substance can be judged to possess inverse agonist
activity to GPRC5D and hence therapeutic activity for
cibophobia. Conversely, provided that the cAMP content (or
the amount of expression of reporter gene) has decreased,
the test substance can be judged to possess agonist activity
to GPC5D and hence therapeutic activity for
lifestyle-related disease.
-
On the other hand, in a screening system containing
phospholipase C (hereinafter also referred to as PLC) as the
effector (i.e., a case wherein the Gα polypeptide contains
the effector interaction region of Gαq), the action of the
Gα polypeptide on the effector can be evaluated by directly
determining the PLC activity. The PLC activity can, for
example, be evaluated by adding 3H-labeled
phosphatidylinositol 4,5-diphosphate to a PLC-containing
membrane fraction, and determining the amount of inositol
phosphate produced using a commonly known technique. The PLC
activity is determined and compared in the presence and
absence of the test substance; provided that the PLC activity
has increased in the presence of the test substance, the test
substance can be judged to possess agonist activity to GPRC5D
and hence therapeutic activity for lifestyle-related disease.
Conversely, provided that the PLC activity has decreased,
the test substance can be judged to possess inverse agonist
activity to GPRC5D and hence therapeutic activity for
cibophobia.
-
When using an intact eukaryotic cell as the screening
system, the action of Gα polypeptide on PLC can also be
evaluated by adding [3H] inositol to the cell and determining
the radioactivity of the resulting [3H] inositol phosphate,
or determining the intracellular Ca2+ content. Although the
intracellular Ca2+ content can be determined by spectroscopy
using a fluorescent probe (fura-2, indo-1, fluo-3,
Calcium-Green I, etc.) or by using a calcium-sensitive
luminescent protein such as aequorin after the cells are
incubated for a given time in the presence and absence of
the test substance, any other commonly known method can be
used. As an apparatus suitable for spectroscopy using a
fluorescent probe, there may be mentioned the FLIPR
(Molecular Devices Company) system.
-
In another embodiment, the Ca2+ content may be
evaluated by determining the amount of expression of reporter
gene under the control of Ca2+-upregulated TPA
(12-O-tetradecanoylphorbol-13-acetate)-respondse element
(TRE). The expression vector used in that method is
described in detail below, and is outlined below. The
intracellular Ca2+ content is determined by culturing a
eukaryotic cell incorporating a vector containing an
expression cassette with a DNA that encodes the reporter
protein linked downstream of a TRE-containing promoter, in
the presence and absence of the test substance for an
appropriate time, disrupting the cells, and measuring and
comparing the expression of the reporter gene in the
thus-obtained extract using a commonly known technique.
-
Accordingly, provided that the intracellular Ca2+
content (or the amount of expression of reporter gene under
the control of TRE) has increased in the presence of a test
substance, the test substance can be judged to possess agonist
activity to GPRC5D and hence therapeutic activity for
lifestyle-related disease. Conversely, provided that the
intracellular Ca2+ content (or the amount of expression of
reporter gene) has decreased, the test substance can be judged
to possess inverse agonist activity to GPRC5D and hence
therapeutic activity for cibophobia.
-
The substance subjected to the screening method of the
present invention may be any commonly known compound or a
new compound, and is exemplified by compound libraries
prepared using combinatorial chemistry techniques, random
peptide libraries prepared by solid phase synthesis or the
phage display method, and naturally occurring components
such as those derived from microorganisms, animals, plants,
and marine organisms.
-
A preferred embodiment of a system containing as
essential constituents a lipid bilayer membrane containing
the GPRC5D protein or an equivalent thereof, and Gα
polypeptide, which system is a constitution unit of the
screening system of the present invention, is a host
eukaryotic cell transfected with both an expression vector
containing a DNA that encodes the GPRC5D protein or an
equivalent thereof and an expression vector containing a DNA
that encodes a polypeptide at least comprising the RB region
of a Gα belonging to a family and the GB region of any Gα,
a homogenate of the cell, or a membrane fraction from the
cell.
-
The "DNA that encodes the GPRC5D protein or an
equivalent thereof" is not particularly limited, as long as
it is a DNA that encodes a polypeptide consisting of the amino
acid sequence shown by SEQ ID NO:2 or SEQ ID NO:4, or a
polypeptide that consists of an amino acid sequence resulting
from the substitution, deletion, insertion, addition or
modification of 1 or more (preferably 1 to 50, more preferably
1 to 30, still more preferably 1 to 10, and most preferably
1 to 5) amino acids in the amino acid sequence shown by SEQ
ID NO:2 or SEQ ID NO:4, that exhibits a ligand-receptor
interaction equivalent to that of GPRC5D, and that couples
with Gα to promote the GDP-GTP exchange reaction of the
subunit or a DNA that encodes an ortholog of a polypeptide
consisting of the amino acid sequence shown by SEQ ID NO:2
or SEQ ID NO:4. As such DNAs, there may be mentioned, for
example the GPRC5D cDNA coding region (the base sequence shown
by base numbers 1-1035 in the base sequence shown by SEQ ID
NO:1 or the base sequence shown by base numbers 148-1047 in
the base sequence shown by SEQ ID NO:3), as well as DNAs that
encodes a GPCR corresponding to GPRC5D of non-human or mouse
mammal origin such as of bovine, swine, simian, or rat; these
can be isolated from cDNA libraries or genomic libraries
derived from cerebral nerve tissue, including the mammal
hypothalamus using the GPRC5D cDNA as a probe. The
equivalent may partially incorporate a mutation introduced
by an artificial treatment such as site-directed mutagenesis
based on the GPRC5D cDNA.
-
The DNAs that encode the three kinds of Gα polypeptides
needs to have at least a sequence that encodes the RB region
of the Gα in each family, and a sequence that encodes the
GB region of any Gα. The sequences of the various Gα genes
are commonly known and the RB region and GB region are well
known from the results of X-ray crystallographic analysis
of Gα as described above. Accordingly, those skilled in the
art can easily construct a fragment lacking a portion of the
coding sequence of Gα as desired.
-
In a screening system based on the action of Gα
polypeptide on the effector as an index, the DNA that encodes
the Gα polypeptide must further contain a nucleic acid
sequence that encodes the effector interaction region.
Because the three kinds of Gα polypeptides share the effector
interaction region as described above, at least two kinds
of Gα polypeptides are chimeras having the effector
interaction region of different families. As the simplest
example of a DNA that encodes the chimeric polypeptide, there
may be mentioned a chimeric polypeptide wherein cDNA sequence
encoding about 5 amino acids at the C-terminus of a Gα
containing the desired effector interaction region have been
replaced with a DNA sequence that encodes the C-terminal
sequence of a Gα belonging to another family.
-
The DNA that encodes the GPRC5D protein or an
equivalent thereof and the DNA that encodes the Gα polypeptide
must be functionally linked to a promoter capable of
exhibiting promoter activity in the host eukaryotic cell.
Any promoter can be used, as long as it is capable of working
in eukaryotic cell; such promoters include, for example,
viral promoters such as the SV40-derived early promoter,
cytomegalovirus LTR, Rous sarcoma virus LTR, MoMuLV-derived
LTR and adenovirus-derived early promoter, and
eukaryote-derived constitutive protein gene promoters such
as the β-actin gene promoter, PGK gene promoter and
transferrin gene promoter. It is preferable that the
expression vector used contain in addition to the
aforementioned promoter a transcription termination signal,
i.e., a terminator region, downstream thereof, and it is
desirable that the expression vector has an appropriate
restriction endonuclease recognition site, preferably a
unique restriction endonuclease recognition site that
cleaves the vector only at one position, so that a coding
DNA can be inserted between the promoter region and the
terminator region. Furthermore, the expression vector may
further contain a selection marker gene (drug resistance
genes such as for tetracycline, ampicillin, kanamycin,
hygromycin and phosphinothricin, auxotrophic mutation
complementary genes, etc.).
-
As examples of vectors useful in the screening system
of the present invention, there may be mentioned plasmid
vectors, viral vectors that are suitable for use in mammals
such as humans, including retrovirus, adenovirus,
adeno-associated virus, herpes virus, vaccinia virus, pox
virus, polio virus, Sindbis virus and Sendai virus, and
vaculovirus vectors that are suitable for use in insect cells.
-
The DNA that encodes the GPRC5D protein or an
equivalent thereof and the DNA that encodes the Gα polypeptide
may be co-transfected to the host cell as carried on two
separate expression vectors, or introduced to the host cell
as inserted in a single vector dicistronically or
monocistronically.
-
The host cell may be any one, as long as it is a
eukaryotic cell such as a mammal cell such as a human, simian,
mouse, rat or hamster cell, or an insect cell. Specifically,
such host cells include mouse-derived cells such as COP, L,
C127, Sp2/0, NS-1, NIH3T3 and ST2, rat-derived cells,
hamster-derived cells such as BHK and CHO, simian-derived
cells such as COS1, COS3, COS7, CV1 and Vero, and
human-derived cells such as HeLa and 293, as well as
insect-derived cells such as Sf9, Sf21 and High Five.
-
Gene introduction to the host cell can be achieved
using any commonly known method applicable to gene
introduction to eukaryotic cells; examples of such methods
include the calcium phosphate co-precipitation method, the
electroporation method, the liposome method, and the
microinjection method.
-
The gene-incorporating host cell can, for example, be
cultured using a minimum essential medium (MEM) containing
about 5% to about 20% bovine fetal serum, Dulbecco's modified
Eagle medium (DMEM), RPMI1640 medium, 199 medium, etc.
Medium pH is preferably about 6 to about 8; culturing
temperature is normally about 27°C to about 40°C.
-
The thus-obtained eukaryotic cell incorporating a DNA
that encodes the GPRC5D protein or an equivalent thereof and
a DNA that encodes a Gα polypeptide may be used as an intact
cell as is, depending on the screening method used, or may
be used in the form of a cell homogenate obtained by disrupting
the cell in an appropriate buffer solution, or a membrane
fraction isolated by centrifuging the homogenate under
appropriate conditions (e.g., supernatant recovered via
centrifugation at about 1,000×g, followed by centrifugation
at about 100,000×g and recovery of the precipitation).
-
For example, when the ligand characteristics of the
test substance are evaluated by GTPγS-binding assay or direct
determination of the effector activity, the screening system
used is preferably a membrane fraction prepared from cells
as described above. On the other hand, when the ligand
characteristics of the test substance are evaluated by
determining the intracellular cAMP content (or the amount
of expression of cAMP-response reporter) or intracellular
Ca2+ content (or the amount of expression of Ca2+-response
reporter), the screening system used is an intact eukaryotic
cell.
-
When evaluating ligand activity with the amount of
expression of a cAMP-responding reporter (in cases where the
effector is adenylate cyclase) or Ca2+-responding reporter
(in cases where the effector is phospholipase C) as an index,
the host eukaryotic cell must incorporate a vector containing
an expression cassette wherein a DNA that encodes the reporter
protein is functionally linked downstream of a promoter
region containing a cAMP-responding element (CRE) or
TPA-responding element (TRE). CRE is a cis-element that
activates gene transcription in the presence of cAMP,
exemplified by a sequence containing TGACGTCA as a consensus
sequence, and may be a sequence containing a deletion,
substitution, insertion or addition, as long as cAMP
responsiveness is retained. On the other hand, TRE is a
cis-element that activates gene transcription in the
presence of Ca2+, exemplified by a sequence containing TGACTCA
as a consensus sequence, and may be a sequence containing
a deletion, substitution, insertion or addition, as long as
Ca2+ responsiveness is retained. As a CRE- or TRE-containing
promoter sequence, there may be used in the same manner virus
promoters and eukaryotic cell constitutive protein gene
promoters as described above; using a restriction enzyme and
DNA ligase, or by means of PCR etc., the CRE or TRE sequence
can be inserted downstream of the promoter sequence. As the
reporter gene under the control of CRE or TRE, there may be
used any commonly known gene that permits quick and simple
detection and quantitation of the expression thereof; such
genes include, for example, but are not limited to, DNAs that
encode such reporter proteins as luciferase, β-galactosidase,
β-glucuronidase, alkaline phosphatase and peroxidase. More
preferably, a terminator sequence is arranged downstream of
the reporter gene. As such a vector carrying a CRE (or
TRE)-reporter expression cassette, there may be used a
commonly known plasmid vector or viral vector.
-
Another preferred example of a system containing as
essential constituents a lipid bilayer membrane containing
the GPRC5D protein or an equivalent thereof, and a Gα
polypeptide, which system is a constitution unit of the
screening system of the present invention, is a host
eukaryotic cell transfected with an expression vector
containing a DNA that encodes a fused protein wherein a
polypeptide at least comprising the RB region of a Gα
belonging to a family and the GB region of any Gα is linked
to the C-terminus side of the GPRC5D protein or an equivalent
thereof, a homogenate of the cell, or a membrane fraction
from the cell.
-
A DNA encoding GPRC5D protein or an equivalent thereof,
and a DNA encoding a polypeptide containing an RB-binding
region of Gα of each family and a GB region of any Gα can
be obtained as mentioned above. Those of ordinary skilled
in the art can easily construct a DNA encoding a fused protein
of GPRC5D and Gα polypeptide by appropriately combining known
genetic engineering methods based on these DNA sequences.
For example, a method comprising ligating a DNA encoding
GPRC5D, whose termination codon has been deleted, with a DNA
encoding GPRC5D to match reading frame using PCR and the like
can be mentioned. In this case, deleting a part of C-terminal
of GPRC5D and inserting a linker sequence such as His-tag
between GPRC5D and Gα are also possible.
-
A DNA encoding the obtained fused protein is inserted
into an expression vector as mentioned above, and introduced
into an eucaryotic host cell by the above-mentioned gene
introduction technique. When the fused protein is expressed
on the obtained eucaryotic cell membrane, and when GPRC5D
and Gα can interact, Gα active domain on intracellular loop
3 of the receptor and RB region of Gα interact in the absence
of a physiological ligand for the receptor, and can promote
the GDP/GTP exchange reaction in Gα. In other words, Gα stays
constitutively being activated. In contrast, a fused
protein with Gα that does not interact with GPRC5D does not
activate GPRC5D, and does not increase Gα-GTP level. Here,
when a GTP analog free of hydrolysis by GTPase activity of
Gα, such as 35S-labeled GTPγS, is added to the system instead
of GTP, activation of GPRC5D can be evaluated by measuring
the radioactivity bound with the membrane and comparing with
each other in the membrane systems respectively containing
three kinds of fused proteins, thereby identifying the Gα
capable of interacting with the receptor.
-
Once a Gα capable of interactng with GPRC5D is
identified, the subsequent screening can be conducted using
only a membrane system containing GPRC5D and the Gα,
preferably only a membrane system containing a fused protein
of GPRC5D and Gα. In other words, in the same manner as in
the identification of the above-mentioned coupled Gα, the
effect of a test substance on the GDP/GTP exchange reaction
in Gα can be evaluated by adding, to the system, a GTP analog
free of hydrolysis due to GTPase activity of Gα, and measuring
and comparing the radioactivities bound with the membrane
in the presence and absence of the test substance, and a
substance having a GPCR ligand activity can be screened for.
When the radioactivity increases in the presence of a test
substance, the test substance has an agonist activity to
GPRC5D, and when the radioactivity decreases, the test
substance has an inverse agonist activity to GPRC5D. Since
a receptor is activated only partially by a fused protein,
when a physiological ligand or an agonist to GPRC5D is bound,
the activity-non-activity balance of the receptor shifts
toward the active side, and the Gα-GTP level increases further.
Thus, this screening system can screen for agonists as well.
-
As shown in the Examples below, because GPRC5D is
constitutively activated only when expressed as a fused
protein with Gas, Gα coupled with the receptor is strongly
suggested to be Gαs. Therefore, the present invention also
provides a screening method for a ligand for the receptor,
which comprises comparing, in a fused protein expression
system of Gαs and the receptor, a GDP/GTP exchange reaction
of Gαs in the presence and absence of the test substance.
-
Activation of GPRC5D in a fused protein can be also
evaluated using, as an index, an action of Gα on an effector.
In this case, the screening system of the present invention
needs to be a membrane system encompassing, in addition to
each fused protein, a lipid bilayer further containing an
effector each Gα interacts with. That is, a membrane system
containing a fused protein with Gαq further contains
phospholipase C (PLC), a membrane system containing a fused
protein with Gαi and Gαs further contains adenylate cyclase
(AC). In this case, the presence or absence of activation
of GPRC5D can be also evaluated by preparing, for each Gα,
a membrane system containing GPRC5D and Gα separately (that
is, not as a fused protein), and measuring and comparing the
activity of effector (that is, in a membrane system containing
a fused protein of GPRC5D and Gα capable of interaction, the
activity of effector is significantly high (low in the case
of Gαi) as compared to a membrane system containing the both
as non-fused proteins, and for those that do not interact,
there is no significant difference in the activity of effector
between the both systems).
-
Once a Gα capable of interacting with GPRC5D is
identified, the subsequent screening can be conducted using
only a membrane system containing a fused protein with said
Gα by directly or indirectly measuring and comparing the
activity of an effector the Gα can interact with, in the
presence and absence of the test substance. Therefore, the
present invention also provides a screening method for a
ligand for GPRC5D, which comprises measuring and comparing,
in a membrane system containing a fused protein of Gα capable
of being coupled with a receptor identified by the
above-mentioned identification method of Gα coupled with
GPRC5D and the receptor and an effector with which said Gα
is capable of interaction, the activity of an effector, in
the presence and absence of the test substance.
-
As mentioned above, because GPRC5D is constitutively
activated only when expressed as a fused protein with Gαs,
Gα coupled with the receptor is strongly suggested to be Gαs.
Therefore, the AC activity in a membrane system containing
a fused protein of GPRC5D and Gαs is measured and compared
in the presence and absence of the test substance. The AC
activity can be measured in the same manner as in the above.
-
It is also known that Gα can be constitutively activated
by introducing a mutation by a known method into a specific
part of a DNA that encodes Gα and modifying the amino acid
sequence thereof. Accordingly, this system can be used
similarly for screening for a ligand. Such technique can be
performed according to the method described in, for example,
Mol. Pharmacol., 57, 890-898 (2000) and Biochemistry, 37,
8253-8261 (1998).
-
For such fused protein (or mutant Gα) expression cell,
too, any form of intact cell, cell homogenate and membrane
fraction can be appropriately selected and used according
to the screening method to be employed.
-
In another embodiment of the present invention, as a
screening system containing, as constituent elements, a
lipid bilayer membrane containing GPRC5D protein or an
equivalent thereof, and Gα polypeptide, one obtained by
re-constituting purified GPRC5D protein or an equivalent
thereof with Gα polypeptide, or a purified fused protein of
the receptor with Gα, in an artificial lipid bilayer membrane
can be used. The GPRC5D protein or an equivalent thereof can
be purified by affinity chromatography with the use of
anti-GPRC5D antibody and the like from membrane fraction
obtained from cerebral nerve tissue and the like including
hypothalamus of human or mouse, or other mammals.
Alternatively, the receptor can be purified by affinity
chromatography using anti-GPRC5D antibody, His-tag, GST-tag
and the like, from a recombinat cell into which an expression
vector containing a DNA encoding GPRC5D protein or an
equivalent thereof has been introduced. Similarly, a fused
protein of the receptor and Gα can be also purified by affinity
chromatography using anti-GPRC5D antibody, His-tag, GST-tag
and the like, from a recombinat cell into which an expression
vector containing a DNA encoding the fused protein has been
introduced.
-
As a lipid composing an artificial lipid bilayer
membrane, phosphatidyl choline (PC), phosphatidyl serine
(PS), cholesterol (Ch), phosphatidyl inositol (PI),
phosphatidyl ethanolamine (PE) and the like can be mentioned.
A mixture of one or more kinds thereof mixed at a suitable
ratio is preferably used.
-
For example, an artificial lipid bilayer membrane
(proteoliposome) incorporating a receptor and Gα or a
receptor-Gα fused protein can be prepared by the following
methods. First, a suitable amount of a mixed lipid
chloroform solution of PC:PI:Ch=12:12:1 is separated in a
glass tube, chloroform is evaporated in a nitrogen gas vapor
to dry the lipid in the form of a film, a suitable buffer
is added to suspend the lipid, which is uniformly dispersed
by ultrasonication, a buffer containing a surfactant such
as sodium cholate and the like is further added to completely
suspend the lipid. Thereto is added a suitable amount of
purified receptor and Gα, or a receptor-Gα fused protein,
and after incubation for about 20-30 min while sometimes
stirring in ice water, dialyzed against a suitable buffer,
centrifuged at about 100,000xg for 30-60 min and the
precipitation is recovered to give a desired proteoliposome.
-
A substance having a therapeutic activity against
cibophobia or a lifestyle-related disease, which is selected
by a screening system or a screening method as mentioned above
can be prepared into a therapeutic agent for cibophobia or
a lifestyle-related disease by combining any
pharmaceutically acceptable carrier.
-
Accordingly, the present invention provides a
therapeutic agent for cibophobia, which comprises as an
active ingredient an antagonist or an inverse agonist to
GPRC5D, which is selected by the screening method of the
present invention. The present invention also provides a
therapeutic agent for a lifestyle-related disease, which
comprises as an active ingredient a physiological ligand or
agonist to GPRC5D, which is selected by the screening method
of the present invention.
-
The pharmaceutically acceptable carrier is exemplified
by, but not limited to, excipients such as sucrose, starch,
mannit, sorbit, lactose, glucose, cellulose, talc, calcium
phosphate, calcium carbonate and the like, binders such as
cellulose, methylcellulose, hydroxypropylcellulose,
polypropylpyrrolidone, gelatine, gum arabic, polyethylene
glycol, sucrose, starch and the like, disintegrating agents
such as starch, carboxymethyl cellulose, hydroxypropyl
starch, sodium-glycol-starch, sodium hydrogen carbonate,
calcium phosphate, calcium citrate and the like, lubricants
such as magnesium stearate, aerosil, talc, sodium lauryl
sulfate and the like, aromatics such as citric acid, menthol,
glycyl lysine ammonium salt, glycine, orange powder and the
like, preservatives such as sodium benzoate, sodium
bisulfite, methylparaben, propylparaben and the like,
stabilizers such as citric acid, sodium citrate, acetic acid
and the like, suspending agents such as methylcellulose,
polyvinylpyrrolidone, aluminum stearate and the like,
dispersing agents such as surfactant and the like, diluents
such as water, physiological saline, orange juice and the
like, base wax such as cacao butter, polyethylene glycol,
refined kerosene and the like, and the like.
-
A preparation which is suitable for oral
administration is, for example, a liquid comprising an
effective amount of a ligand dissolved in a diluent such as
water, physiological saline and orange juice, a capsule,
sachet or tablet comprising an effective amount of a ligand
as a solid or granules, a suspension comprising an effective
amount of a ligand in a suitable dispersion medium, an
emulsion comprising a solution of an effective amount of a
ligand dispersed and emulsified in a suitable dispersion
medium and the like.
-
A preparation preferable for parenteral administration
(e.g., subcutaneous injection, intramuscular injection,
topical injection, intraperitoneal administration and the
like) includes, for example, an aqueous or non-aqueous
isotonic sterile injection which may contain antioxidant,
buffer, bacteriostatic agent, isotonicity agent and the like.
It may be an aqueous or non-aqueous sterile suspension which
may contain suspension, solubilizer, thickener, stabilizer,
preservative and the like. When the administration method
is topical injection near the hypothalamus, an injection
containing ligand as an active ingredient dissolved or
suspended in an artificial cerebrospinal fluid is preferable.
Alternatively, it can be formulated into a sustained release
preparation using a biocompatible material such as collagen
and the like. Since pluronic gel gelates at body temperature
and becomes a liquid at a lower temperature, long duration
can be afforded by topically injecting the ligand along with
pluronic gel to allow for gelation around the target tissue.
The ligand preparation can be sealed in a container in a unit
dose or plural doses like an ampoule or vial. It is also
possible to lyophilize a ligand and a pharmaceutically
acceptable carrier and preserve them in a state that only
requires dissolving or suspending in a suitable sterile
vehicle immediately before use.
-
While the dose of the ligand preparation of the present
invention varies depending on ligand activity (full agonist
or partial agonist, or an antagonist or inverse agonist),
degree of seriousness of the disease, the animal species to
be the administration subject, drug acceptability, body
weight and age of the administration subject, and the like,
it is generally about 0.0008 - about 2.5 mg/kg, preferably
about 0.008 - about 0.025 mg/kg, a day for an adult in the
amount of the ligand.
-
The present invention is explained in more detail by
referring to Examples, which are mere exemplification and not to
be construed as limitative. Unless particularly specified, the
following examples were performed according to the methods
described in Sambrook, J. et al., Molecular Cloning: A
Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory
Press, New York (1989), Current Protocols in Protein Science
(ed. Coligan, J. E. et al.), John Wiley and Sons, Inc. and
the like.
Reference example 1: Analysis of expression distribution of
GPRCSD gene by RT-PCR
-
The hypothalamus from db/db mouse (male, SPF grade,
7-week-old at the time of purchase, 10-week-old at the time
of organ sampling, CLEA JAPAN, INC.) was homogenized in Trizol
(Gibco), and total RNA was obtained. cDNA was synthesized
using RNA PCR Kit (AMV) Ver2.1. (Takara) and the total RNA
(1 µg) as a template. PCR was performed using the obtained
cDNA from hypothalamus as a template and the following mouse
GPRC5D (mGprc5d) specific primer. As a Taq polymerase, one
from Perkin Elmer, Inc. was used.
As a result, expression of GPRC5D gene in the hypothalamus
of db/db mouse was clearly shown.
Reference example 2: Analysis of expression distribution of
GPRC5D gene by in situ hybridization
-
After perfusion fixation with 10% neutral buffered
formalin, the brain was taken from mouse (CD-1 (ICR)) and after
fixation with 10% neutral buffered formalin, the block was
prepared by paraffin embedding. The paraffin block was
sliced in a 7µm-thick coronal section (in the vicinity of
Interaural 2.34 mm, Bregma-1.46 mm), and used as a section
for ISH staining.
-
In addition, digoxigenin-labeled RNA probe was
prepared by an In vitro transcription method with T3 and T7RNA
polymerase. Specifically, antisense RNA probe
corresponding to the region of 234th - 624th of SEQ ID NO: 3
was prepared using DIG RNA Labeling Mix (Roche).
-
Next, using mouse brain paraffin section and probe
prepared above, ISH staining was performed by a conventional
method. Using anti-digoxigenin antibody labeled with
alkaline phosphatase as antibody and NBT/BCIP as coloring
substrate, nuclear staining with Kernechtrot was performed
after staining.
-
As a result of staining using a GPRC5D antisense probe,
expression in nucleus paraventricularis and hypothalamus
ventromedial nucleus was found. The nucleus
paraventricularis and hypothalamus ventromedial nucleus are
the regions where the feeding center exists. Thus, GPRC5D
was suggested to be a factor involved in the feeding and energy
metabolism in the feeding center.
Example 1: Action of GPRC5D clone antisense DNA on obese model
mouse
(1) Experimental materials
-
Reagent: As a GPRC5D clone antisense, a 25 mer thiolized
antisense DNA was used, which corresponded to the vicinity
of initiation codon of the gene. The base sequences of the
antisense DNA are shown in the following.
-
As a control DNA, thiolized oligo DNA having the
following sequence was used.
-
This sequence is complimentary to the sequence produced
by the mutation which causes a splicing abnormality at
position 705 in pre-mRNA of erythrocyte β-globin in
hemoglobinopathy thalassemia. It is assumed that the
oligosaccharide has no specific target region or biological
activities in normal cells and corrects the splicing
abnormality only when acted on erythrocyte of thalassemia,
thereby producing normal β-globin coding mRNA. The
synthesis, thiolation and HPLC purification of these oligo
DNAs were committed to Nihon Bio Service Co., Ltd.
-
For other reagents, commercially available special
grade reagents were used.
- Experimental animal: C57BL db/db (hereinafter, referred as
obese mouse) (SPF grade) were purchased from CLEA JAPAN, INC.,
and after preliminary breeding, the obese mice were used for
the test at the age of 10 weeks.
- Breeding environment: The mice were bred in a room controlled
to a temperature of not lower than 20°C and not higher than
26°C, relative humidity of not less than 30% and not more than
70%, lighting cycle of 8:00-20:00 lighting and 20:00-8:00
lights-out. During breeding, the mice were allowed a free
access to a solid feed (CE-2, CLEA JAPAN) and sterile
distilled water.
-
(2) Preparation of administration liquid
-
As an antisense DNA administration liquid and a control
DNA administration liquid, an artificial cerebrospinal fluid
(0.166 g/L CaCl2, 7.014 g/L NaCl, 0.298 g/L KCl, 0.203 g/L
MgCl2·6H2O, 2.10 g/L NaHCO3) containing 7.5 µg/µl of antisense
DNA was prepared.
(3) Administration of antisense DNA in cerebral ventricle
-
The obese mice were divided into two groups, and after
fasting overnight, an antisense DNA administration liquid
was given at 4 µl/mouse (30 µg/mouse for antisense DNA) to
one group and a control DNA administration liquid (4 µl/mouse)
was administered into the lateral ventricle of the other group,
simultaneously with the lighting at 8:00 a.m..
(4) Effect of antisense DNA administration on food
consumption and blood glucose level of mice
-
Feeding of mice was resumed immediately after
administration, and food consumption was calculated every
12 hours up to 48 hours after administration. The effect of
the antisense DNA on food consumption of obese mice is shown
in Fig. 1. In Fig.1, black columns show average food
consumption at every 12 hours after administration to control
DNA administration group and white columns show that of
antisense DNA administration group (error bar is standard
deviation, control DNA administration groups n=4, antisense
DNA administration groups n=4). There was no big change in
the amount of food consumption up to 12 hours after
administration. As the time advances, however,
administration of the antisense DNA of GPRC5D clone showed
an effect of increased food consumption in obese mice.
-
Additionally, blood glucose level was measured before
administration and every 24 hours up to 48 hours after
administration. The effect of antisense DNA on blood glucose
level of obese mice is shown in Fig. 2. In Fig.2, black
columns show average blood glucose level at every 24 hours
after administration of control DNA administration groups
and white columns show that of antisense DNA administration
groups (error bar is standard deviation, control DNA
administration groups n=4, antisense DNA administration
groups n=4). There was no big change in the blood glucose
level up to 24 hours after administration. At 48 hours after
administration, however, administration of the antisense DNA
of GPRC5D clone showed an effect of increased blood glucose
level in obese mice.
-
From the above-mentioned results, the possibility was
suggested that GPRC5D clone is a factor involved in feeding
behavior and glucose metabolism.
Example 2: Analysis of variation in hypothalamus gene
expression in satiation and fasting states
-
Hypothalamus was taken from db/db mouse (male, SPF grade,
7-week-old at the time of purchase, 11-week-old at the time
of organ sampling, CLEA JAPAN, INC.; hereinafter to be
referred to as obese mouse) and C57BL/6N mouse (male, SPF
grade,
6-week-old at the time of purchase, 11-week-old at the time
of organ sampling, Charles River, Japan, Inc.; hereinafter
to be reffered to as nomal mouse) in satiation state or 24
hour fasting state, homogenated in Trizol (Gibco) and total
RNA was obtained. Reverse transcription reaction was
performed using 1 µg of total RNA and SYBR Green RT-PCR Reagent
(Roche). Subsequently, each sample after reverse
transcription reaction and GPRC5D clone specific primer (see
below) were mixed, PCR reaction was performed using ABI7700
sequence detector (PE biosystems) and variation in the
expression of gene of GPRC5D clone was analyzed. In addition,
GPRC5D clone expression was calibrated with GAPDH
(glyceraldehyde-3-phosphate dehydrogenase) and analyzed as
relative values.
·GPRC5D
-
·GAPDH
-
-
Variation in expression of GPRC5D gene in the
satiation/fasting state is shown in Fig. 3. In Fig. 3, black
columns show average variation in the expression of GPRC5D
gene in satiation state, white columns show that in the
expression of GPRC5D gene in the fasting state (error bar
shows standard deviation, n=3 in each groups). There was
found no big variation in normal mouse. On the contrary,
promoted expression of GPRC5D gene was found in obese mouse
in the fasting state. The promoted expression of GPRC5D
clone in obese mouse in the fasting state and promoted food
consumption and increased blood glucose level due to the
administration of antisense suggested the possibility of
GPRC5D clone having a food consumption suppressive action.
From the above findings, it has been clarified that GPRC5D
clone is involved in the regulation of food consumption and
glucose metabolism, and that promotion of the action of GPRC5D
clone provides a more effective therapeutic effect on
lifestyle-related diseases.
Example 3: Establishment of GPRC5D stable expression cell
line
(1) Construction of transgene vector
-
The coding region of GPRC5D clone is amplified by PCR
method using KOD-Plus (TOYOBO). The amplified gene fragment
is inserted into pcDNA3.1 (Invitrogen) and a GPRC5D
expression vector is constructed.
(2) Establishment of cell line
-
CHO cell lines expressing three types of chimeric G
proteins (Gq, Gqi5, Gqs5) are seeded in 10 cm2 culture dishes
and cultured in a D-MEM medium (Gibco) containing 10% FBS
(Gibco) until 60-70% confluent. Then, the cells are
transferred to a serum-free medium, complexed with the GPRC5D
expression vector constructed in the above (1) and
Lipoofectamine-Plus (Gibco), and added to the medium. After
incubation for 5 hours, the medium is changed to D-MEM medium
containing 10% FBS and the cells are further cultured for
8 hours. Then cells are detached from the petri dish with
trypsin-EDTA, suspended in D-MEM medium containing 500 µg/ml
G418 and 10% FBS and seeded in 10 cm2 petri dishes. Few days
later, formed colonies are isolated and named as GPRC5D stable
expression cell lines (¥q, ¥qi5, ¥qs5).
Example 4: Screening for receptor agonist
(1) Preparation of cells
-
GPRC5D stable expression cell lines (¥q, ¥qi5, ¥qs5)
are seeded in 96-well culture dishes and cultured in a D-MEM
medium (Gibco) containing 10% FBS (Gibco) until 60-70%
confluent. They are used as expression cells.
(2) Addition of reagents
-
The medium of expression cells is changed to serum free
D-MEM medium one day before use. On assessment day, each
compound (2.5 mM DMSO solution) is diluted to desired
concentrations with D-MEM medium. Medium in the culture
dishes is removed, and the diluted test compound, 4 µM
Fluo3-AM (Teflab.) and 2.5 mM probenecid are added and it
is cultured at 37°C for 60 min. A sample treated in the same
way except that the test compound is not added is prepared
for comparison.
(3) Measurement of intracellular calcium concentrations by
FLIPR method
-
The cells treated as mentioned above are washed with
ice-cooled PBS, and suspended in Thyrode's medium
(containing 2.5 mM probenecid and 1% gelatin). Absorbance
of culture dish at 488 nM and 540 nM is quantified by FLIPR
(Molecular Devices).
Example 5: Construction of plasmid for expression of
GPRC5D-Gα fused protein
-
As three kinds of representative α subunits considered
to almost cover all GPCR signaling, Gα16 was selected from
the Gq family, Gαi2 was selected from the Gi family and GαS2
was selected from the Gs family. All these coding regions
were PCR cloned into expression vector pcDNA3.1(+). A
restriction enzyme EcoRV cleavage site and a sequence
containing 6×His tags were added just before each Gα coding
region by PCR, whereby plasmids pcHISGα16, pcHISGαi2 and
pcHISGαS2 capable of easy fusion with GPCR gene on the 5'
side of each Gα protein were prepared.
-
That is, human spleen cDNA (Clontech) was diluted 20
times, 1 µl thereof was used as a template for amplification.
As the enzyme, employed was KODplus (TOYOBO). However, the
Mg2+ concentration was 1 mM. For amplification reaction, 20
µl of a reaction solution was used, which was a reaction
buffer attached with KODplus. To amplify Gα16 cDNA, primer
GNA15F1 (SEQ ID NO: 13) and GNA15R1 (SEQ ID NO: 14) were
used; to amplify Gαi2 cDNA, primer GNAi2F1 (SEQ ID NO: 15)
and GNAi2R1 (SEQ ID NO: 16) were used; and to amplify GaS2
cDNA, primer GS2F1 (SEQ ID NO: 17) and GS2R1 (SEQ ID NO:
18) were used. For amplification, GeneAmp PCR System 9600
(Applied Biosystems) was used and amplification was
conducted under the conditions of 94°C, 2 min → (94°C, 15
sec → 68°C, 120 sec) × 40 cycles to give a PCR product having
an object size. The terminal of each PCR product was
phosphorylated, separated and purified by 0.8% agarose gel
electrophoresis, cleaved with restriction enzyme EcoRV,
ligated with plasmid pcDNA3.1(+)(Invitrogen) treated with
CIAP and used to transform Escherichia coli DH5α. Plasmids
were purified from the transformant line, and upon
confirmation that the restriction enzyme digestion pattern
and inserted base sequence were objective ones, the obtained
G protein expression plasmids were named pcGα16, pcGαi2 and
pcGaS2, respectively.
-
To fuse GPRC5D via HIS tag on N terminal of each G
protein, HIS tag sequence was ligated immediately before
initiation codon ATG of each G protein, and a restriction
enzyme EcoRV recognition site was further added. That is,
PCR was conducted using 10 ng each of pcGα16, pcGαi2 and
pcGaS2 as a template, a primer (GNA15ATG; SEQ ID NO: 19,
GNAi2ATG; SEQ ID NO: 20 or GS2ATG; SEQ ID NO: 21) and a primer
(pcDNARV; SEQ ID NO: 22) of vector. Amplification was
conducted under the conditions of 94°C, 2 min → (94°C, 15
sec → 58°C, 30 sec → 68°C, 60 sec) × 20 cycles to give a
PCR product having the object size. Then HIS2 linker (SEQ
ID NO: 23) and HIS2 linker (R) (SEQ ID NO: 24) were annealed,
and after phosphorylation of the terminal, ligated with each
PCR product. These were digested with restriction enzymes
EcoRV and XhoI, and the object DNA fragments were separated
and purified by 0.8% agarose gel electrophoresis. The
recovered DNA fragments were ligated with expression
plasmid pcDNA3.1 (+) digested with EcoRV and XhoI, and used
to transform Escherichia coli DH5α. Plasmids were purified
from transformant line, and upon confirmation that the
restriction enzyme digestion pattern and inserted base
sequence were object ones, the obtained plasmids were named
pcHISGα16, pcHISGαi2 and pcHISGαS2, respectively.
Example 6: Construction of GPRC5D-Gα fused protein
expression plasmid
(1) cloning of GPRC5D
-
PCR cloning of GPRC5D gene was performed with human
testis derived cDNA library (clontech) and the following
primers.
-
The obtained fragment was subjected to TA-cloning with
pT7Blue vector (Novagen). The plasmid was prepared from the
obtained clone, and analyzed for the base sequence. As a
result, GPRC5D gene having a sequence identical to the
sequences of published references was obtained. The
obtained plasmid was named as pT7GPRC5D.
(2) construction of GPRC5D expression vector
-
Construction of expression vector was carried out using
GATEWAY system of Invitrogen Corp. and in accordance with
the vendor's instructions. First, expression vectors were
converted to destination vectors. pcHISGαS2, pcHISGαi2 and
pcHISGα16 prepared in example 5 were each digested with EcoRV
then GATEWAY frame B (Invitrogen) was inserted. DB3.1
compitent cells (Invitrogen) after transformation were
selected with chloramphenicol, the obtained clones were
further selected by digesting with restriction enzymes to
give desired clones having GATEWAY frame B insert in the right
direction. These were named as pcHisGαS2-DEST,
pcHisGαi2-DEST and pcHisGα16-DEST, respectively. In
addition, a destination vector having the same CMV promoter
and bGH terminator was used as a non-fused gene expression
vector for control. Specifically, pcDNA3.1mycHisA was
digested with Hind III-Xba I and then blunted, and GATEWAY
frame C.1 (Invitrogen) was inserted. Then DB3.1 compitent
cells (Invitrogen) after transformation were selected with
chloramphenicol, the obtained clones were further selected
by digesting with restriction enzymes, and a clone having
GATEWAY frame C.1 insert in the right direction was obtained,
which was named as pcDNA3.1-DEST.
-
Next, using pT7GPRC5D as a template and the following
primers, ORF (R-type) having up to intact stop codon, and
ORF (F-type) with only intact stop codon deleted were
amplified by PCR. After electrophoresis and purification by
cutting out from the gel, these ORFs were subjected to BP
clonase (Invitrogen) reaction to be newly carried on donor
vector pDONR201.
-
By analysis of the base sequence of the obtained clones,
desired clones were each confirmed to have been obtained.
The obtained clones were named as pENTR/GPRC5D/R (R-type
entry clone) and pENTR/GPRC5D/F (F-type entry clone),
respectively.
-
By LR clonase reaction of these, pENTR/GPRC5D/F was
crossed over 3 kinds of Gα fused destination vectors
(pcHisGαS2-DEST, pcHisGαi2-DEST and pcHisGα16-DEST) and
pENTR/GPRC5D/R was crossed over pcDNA3.1-DEST. In these
obtained clones, plasmids were prepared on a small scale and
the desired clone was selected by restriction enzyme
treatment. The obtained plasmids were named as
pcC5D/His/GαS2, pcC5D/His/Gαi2, pcC5D/His/Gα16 and pcC5D,
respectively. Further, using Qiagen Maxi Kit, mass
preparation and purification of plasmid were done from 100
ml of culture fluid with the aim of introduction into cell
lines.
Example 7: Confirmation of constitutive activation by
expression of GPRC5D-Gα fused protein
-
To examine whether constitutive activation occurs by
cellular expression a protein with Gα protein fused at the
c-terminal of GPRC5D, the following experiment was carried
out.
-
1 µg of DNA for four kinds of plasmid vectors (pcC5D,
pcC5D/His/GαS2, pcC5D/His/Gαi2 and pcC5D/His/Gα16)
constructed in Example 6, and pcDNA3.1 (+) (Invitrogen: Cat.
No. V790-20) were diluted with 125 µl of OPTI-MEM I medium
(Invitrogen: Cat. No. 31985-062) (solution A). 2.5 µl of
Lipofectamine 2000 Reagent (Invitrogen: Cat. No. 1168-027)
which is a transfection reagent, were diluted with 125 µl
of OPTI-MEM I medium and stood for 5 minutes (solution B).
Solution A and solution B were mixed and incubated for 20
minutes, and then added at 50 µg/well (triplicate) to Chinese
Hamster Ovary cells (CHO-K1 cells, ATCC No. CCL-61) plated
3 x 104 cells/well the day before. After culture at 37°C for
4 hours under condition of 5% CO2, the medium was changed to
F12 medium (Invitrogen: Cat. No. 11765-054) containing 10%
FCS at 100 µl/well and further cultured for about 12 hours.
Intracellular cAMP levels of these cells were measured using
HitHunter™ EFC Cyclic AMP Chemiluminescens Assay Kit
(Applied Biosystem: Cat. No. DRX-0027), cAMP measurement kit,
and in accordance with the attached protocol (Fig. 4). As
a result, cAMP levels were confirmed to have been specifically
increased by fusing GPRC5D with GαS2, suggesting that GPRC5D
can be constitutively activated by fusing with GαS2.
Therefore, the GPRC5D was strongly suggested to be a GPCR
that is coupled with GαS.
Sequence Listing Free Text
-
- SEQ ID NO: 5: Oligonucleotide designed to function as a primer
to amplify mRNA of GPRC5D.
- SEQ ID NO: 6: Oligonucleotide designed to function as a primer
to amplify mRNA of GPRC5D.
- SEQ ID NO: 7: Oligonucleotide designed to function as an
antisense DNA inhibiting expression of GPRC5D.
- SEQ ID NO: 8: Oligonucleotide designed to function as an
antisense DNA for sequence resulted from mutation causing
abnormal splicing at position 705 of β-globin pre-mRNA in
thalassemia.
- SEQ ID NO: 9: Oligonucleotide designed to function as a
primer to amplify mRNA of GPRC5D.
- SEQ ID NO: 10: Oligonucleotide designed to function as a
primer to amplify mRNA of GPRC5D.
- SEQ ID NO: 11: Oligonucleotide designed to function as a
primer to amplify mRNA of GAPDH.
- SEQ ID NO: 12: Oligonucleotide designed to function as a
primer to amplify mRNA of GAPDH.
- SEQ ID NO: 13: Oligonucleotide designed to function as a sense
primer to amplify human G protein Gα16 cDNA fragment
containing full length ORF.
- SEQ ID NO: 14: Oligonucleotide designed to function as an
antisense primer to amplify human G protein Gα16 cDNA
fragment containing full length ORF.
- SEQ ID NO: 15: Oligonucleotide designed to function as a
sense primer to amplify human G protein Gαi2 cDNA fragment
containing full length ORF.
- SEQ ID NO: 16: Oligonucleotide designed to function as an
antisense primer to amplify human G protein Gαi2 cDNA
fragment containing full length ORF.
- SEQ ID NO: 17: Oligonucleotide designed to function as a
sense primer to amplify human G protein GαS2 cDNA fragment
containing full length ORF.
- SEQ ID NO: 18: Oligonucleotide designed to function as an
antisense primer to amplify human G protein GαS2 cDNA
fragment containing full length ORF.
- SEQ ID NO: 19: Oligonucleotide designed to function as a sense
primer to amplify human G protein Gα16 cDNA fragment from
the initiation codon.
- SEQ ID NO: 20: Oligonucleotide designed to function as a sense
primer to amplify human G protein Gαi2 cDNA fragment from
the initiation codon.
- SEQ ID NO: 21: Oligonucleotide designed to function as a sense
primer to amplify human G protein GαS2 cDNA fragment from
the initiation codon.
- SEQ ID NO: 22: Oligonucleotide designed to function as an
antisense primer to amplify multicloning sites of plasmid
pcDNA3.1(+).
- SEQ ID NO: 23: Sense chain oligonucleotide designed to
construct a linker containing a nucleotide sequence encoding
a 6xHis tag peptide sequence.
- SEQ ID NO: 24: Antisense chain oligonucleotide designed to
construct a linker containing a nucleotide sequence encoding
6xHis tag peptide sequence.
- SEQ ID NO: 25: Oligonucleotide designed to function as a sense
primer to amplify cDNA of GPRC5D.
- SEQ ID NO: 26: Oligonucleotide designed to function as an
antisense primer to amplify cDNA of GPRC5D.
- SEQ ID NO: 27: Oligonucleotide designed to function as a sense
primer to amplify ORF of cDNA of GPRC5D.
- SEQ ID NO: 28: Oligonucleotide designed to function as an
antisense primer to amplify ORF(R-type) of cDNA of GPRC5D.
- SEQ ID NO: 29: Oligonucleotide designed to function as an
antisense primer to amplify ORF(F-type) of cDNA of GPRC5D.
-
Industrial Applicability
-
Since GPRC5D is a GPCR involved in feeding behavior,
the pharmaceutical composition of the present invention
containing, as an active ingredient, a substance that
enhances or suppresses expression or function of GPRC5D can
regulate food intake to a desired level and is expected to
afford a therapeutic effect on lifestyle-related diseases
caused by overeating, such as diabetes, obesity,
hyperlipidemia and the like, or cibophobia. According to the
screening system and screening method of the present
invention, moreover, a ligand for GPRC5D can be easily and
rapidly screened for and they are useful for the development
of a new drug targeting GPRC5D, search for a disease marker
and establishment of a diagnostic method using the disease
marker.
-
While the present invention has been described with an
emphasis on preferred embodiments, it will be obvious to those
of ordinary skilled in the art that variations of the preferred
embodiments may be used. It is intended that the invention
may be practiced otherwise than as specifically described
herein. Accordingly, this invention includes all
modifications encompassed within the spirit and scope of the
invention as defined by the following claims.
-
This application is based on a patent application No.
397523/2001 filed in Japan, the contents of which are hereby
incorporated by reference. The references cited herein,
including patents and patent applications, are hereby
incorporated in their entireties by reference, to the extent
that they have been disclosed herein.